Antigen binding peptides (abtides) from peptide libraries

ABSTRACT

Abtides are provided. Abtides are peptides identified by a two-step process of screening random peptide libraries. In the first step, the target ligand is an antibody or receptor (or derivative thereof). The peptides identified in the first screening step are used as target ligands in the second screening step. The peptides identified in the second screening step are abtides. Abtides possess binding specificities that are similar to the binding specificities of the antibodies or receptors that are used in the first screening step. Abtides may be used in place of antibodies in many assays or therapeutic applications. 
     Abtides binding to polymorphic epithelial mucin (PEM) are provided. 
     Also provided are methods of obtaining abtides as well as diagnostic and therapeutic compounds containing abtides.

This application is a division of U.S. patent application Ser. No.08/488,161 filed Jun. 7, 1995, which issued as U.S. Pat. No. 5,885,577on Mar. 23, 1999, which in turn is a continuation-in-part of U.S. patentapplication Ser. No. 08/310,192 filed Sep. 21, 1994 now abandoned, theentire contents of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

The present invention relates generally to peptides capable of specificbinding to ligands of interest. The present invention also relates topeptides capable of mimicking the specific binding of a receptor to itsligand, an antibody to its antigen, and the like. Such peptides areknown as "abtides." Abtides are identified by first and second screeningsteps of peptide libraries. The first screening step uses an antibody orreceptor as a first target ligand and identifies peptide sequences("mimetopes") which specifically bind to the antibody or receptor. Themimetopes are then incorporated into a second target ligand in a secondscreening step to identify abtides that bind the mimetope. Abtides mimicthe binding specificity of the antibody (to its antigen) or the receptor(to its ligand) that was used as the first target ligand in the firstscreening step. The invention further relates to the use of abtides inthe place of antibodies in assays. The invention also provides abtidecompositions for use in therapy and diagnosis of disease.

2. BACKGROUND OF THE INVENTION 2.1. Peptide Libraries

The use of peptide libraries is well known in the art. Such peptidelibraries have generally been constructed by one of two approaches.According to one approach, peptides have been chemically synthesized invitro in several formats. For example, Fodor et al., 1991, Science 251:767-773, describes use of complex instrumentation, photochemistry andcomputerized inventory control to synthesize a known array of shortpeptides on an individual microscopic slide. Houghten et al., 1991,Nature 354: 84-86, describes mixtures of free hexapeptides in which thefirst and second residues in each peptide were individually andspecifically defined. Lam et al., 1991, Nature 354: 82-84, describes a"one bead, one peptide" approach in which a solid phase split synthesisscheme produced a library of peptides in which each bead in thecollection had immobilized thereon a single, random sequence of aminoacid residues. For the most part, the chemical synthetic systems havebeen directed to generation of arrays of short length peptides,generally fewer than about 10 amino acids or so, more particularly about6-8 amino acids. Direct amino acid sequencing, alone or in combinationwith complex record keeping of the peptide synthesis schemes, isrequired to use these libraries.

According to a second approach using recombinant DNA techniques,peptides have been expressed in biological systems as either solublefusion proteins or viral capsid fusion proteins.

A number of peptide libraries according to the second approach have usedthe M13 phage. M13 is a filamentous bacteriophage that has been aworkhorse in molecular biology laboratories for the past 20 years. M13viral particles consist of six different capsid proteins and one copy ofthe viral genome, as a single-stranded circular DNA molecule. Once theM13 DNA has been introduced into a host cell such as E. coli, it isconverted into double-stranded, circular DNA. The viral DNA carries asecond origin of replication that is used to generate thesingle-stranded DNA found in the viral particles. During viralmorphogenesis, there is an ordered assembly of the single-stranded DNAand the viral proteins, and the viral particles are extruded from cellsin a process much like secretion. The M13 virus is neither lysogenic norlytic like other bacteriophage (e.g., λ); cells, once infected,chronically release virus. This feature leads to high titers of virus ininfected cultures, i.e., 10¹² pfu/ml.

The genome of the M13 phage is ˜8000 nucleotides in length and has beencompletely sequenced. The viral capsid protein, protein III (pIII) isresponsible for infection of bacteria. In E. coli, the pillin proteinencoded by the F factor interacts with pIII protein and is responsiblefor phage uptake. Hence, all E. coli hosts for M13 virus are consideredmale because they carry the F factor. Several investigators havedetermined from mutational analysis that the 406 amino acid long pIIIcapsid protein has two domains. The C-terminus anchors the protein tothe viral coat, while portions of the N-terminus of pIII are essentialfor interaction with the E. coli pillin protein (Crissman and Smith,1984, Virology 132: 445-455). Although the N-terminus of the pIIIprotein has been shown to be necessary for viral infection, the extremeN-terminus of the mature protein does tolerate alterations. In 1985,George Smith published experiments reporting the use of the pIII proteinof bacteriophage M13 as an experimental system for expressing aheterologous protein on the viral coat surface (Smith, 1985, Science228: 1315-1317). It was later recognized, independently by two groups,that the M13 phage pIII gene display system could be a useful one formapping antibody epitopes. De la Cruz et al., 1988, J. Biol. Chem. 263:4318-4322 cloned and expressed segments of the cDNA encoding thePlasmodium falciparum surface coat protein into the pIII gene, andrecombinant phage were tested for immunoreactivity with a polyclonalantibody. Parmley and Smith, 1988, Gene 73: 305-318 cloned and expressedsegments of the E. coli β-galactosidase gene in the pIII gene andidentified recombinants carrying the epitope of an anti-β-galactosidasemonoclonal antibody. The latter authors also described a process termed"biopanning", in which mixtures of recombinant phage were incubated withbiotinylated monoclonal antibodies, and phage-antibody complexes couldbe specifically recovered with streptavidin-coated plastic plates.

In 1989, Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218suggested that short, synthetic DNA segments cloned into the pIII genemight represent a library of epitopes. These authors reasoned that sincelinear epitopes were often ˜6 amino acids in length, it should bepossible to use a random recombinant DNA library to express all possiblehexapeptides to isolate epitopes that bind to antibodies.

Scott and Smith, 1990, Science 249:386-390 describe construction andexpression of an "epitope library" of hexapeptides on the surface ofM13. The library was made by inserting a 33 base pair Bgl I digestedoligonucleotide sequence into an Sfi I digested phage fd-tet, i.e.,fUSE5 RF. The 33 base pair fragment contains a random or "degenerate"coding sequence (NNK)₆ where N represents G, A, T or C and K representsG or T. The authors stated that the library consisted of 2×10⁸recombinants expressing 4×10⁷ different hexapeptides; theoretically,this library expressed 69% of the 6.4×10⁷ possible peptides (206).Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382 alsodescribed a somewhat similar library of hexapeptides expressed as pIIIgene fusions of M13 fd phage. PCT publication WO 91/19818 dated Dec. 26,1991 by Dower and Cwirla describes a similar library of pentameric tooctameric random amino acid sequences.

Devlin et al., 1990, Science, 249:404-406, describes a peptide libraryof about 15 residues generated using an (NNS) coding scheme foroligonucleotide synthesis in which S is G or C.

Christian and colleagues have described a phage display library,expressing decapeptides (Christian et al., 1992, J. Mol. Biol.227:711-718). The starting DNA was generated by means of anoligonucleotide comprising the degenerate codons [NN(G/T)]₁₀ with aself-complementary 3' terminus. This sequence, in forming a hairpin,creates a self-priming replication site which could be used by T4 DNApolymerase to generate the complementary strand. The double-stranded DNAwas cleaved at the Sfi I sites at the 5' terminus and hairpin forcloning into the fUSE5 vector described by Scott and Smith, supra.

Other investigators have used other viral capsid proteins for expressionof non-viral DNA on the surface of phage particles. The protein pVIII isa major M13 viral capsid protein and interacts with the single strandedDNA of M13 viral particles at its C-terminus. It is 50 amino acids longand exists in approximately 2,700 copies per particle. The N-terminus ofthe protein is exposed and will tolerate insertions, although largeinserts have been reported to disrupt the assembly of pVIII fusionproteins into viral particles (Cesareni, 1992, FEBS Lett. 307:66-70). Tominimize the negative effect of pVIII fusion proteins, a phagemid systemhas been utilized. Bacterial cells carrying the phagemid are infectedwith helper phage and secrete viral particles that have a mixture ofboth wild-type and pVIII fusion capsid molecules. pVIII has also servedas a site for expressing peptides on the surface of M13 viral particles.Four and six amino acid sequences corresponding to different segments ofthe Plasmodium falciparum surface antigen have been cloned and expressedin the comparable gene of the filamentous bacteriophage fd (Greenwood etal., 1991, J. Mol. Biol. 220:821-827).

Lenstra, 1992, J. Immunol. Meth. 152:149-157 described construction of alibrary by a laborious process encompassing annealing oligonucleotidesof about 17 or 23 degenerate bases with an 8 nucleotide long palindromicsequence at their 3' ends. This resulted in the expression of randomhexa- or octa-peptides as fusion proteins with the β-galactosidaseprotein in a bacterial expression vector. The DNA was then convertedinto a double-stranded form with Klenow DNA polymerase, blunt-endligated into a vector, and then released as Hind III fragments. Thesefragments were then cloned into an expression vector at the C-terminusof a truncated β-galactosidase to generate 10⁷ recombinants. Colonieswere then lysed, blotted on nitrocellulose filters (10⁴ /filter) andscreened for immunoreactivity with several different monoclonalantibodies. A number of clones were isolated by repeated rounds ofscreening and were sequenced.

Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869 described asystem in which random peptides were fused to the carboxy terminus ofthe lac repressor. The fusion proteins contained an intact lac aminoterminus (which is responsible for specific binding of the lac repressorto the DNA sequences constituting the lac operator sites). Thenucleotide sequences encoding the fusion protein were cloned into aplasmid containing copies of the lac operator site. Thus, when thefusion protein was expressed in bacteria, it became bound to theoperator sites of the plasmid encoding it. This provided a physicallinkage between the fusion protein and the gene encoding it. Whenbacteria containing the plasmid were screened with ligands for which itwas desired to isolate binding partners, the fusion proteins comprisingpeptides that specifically bound to the ligand were isolated, carryingalong the genes that encoded those fusion proteins.

A comprehensive review of various types of peptide libraries can befound in Gallop et al., 1994, J. Med. Chem. 37:1233-1251.

2.2. Ligands Used to Screen Peptide Libraries

Screening of peptide libraries has generally been confined to the use ofa restricted number of ligands. Most commonly, the ligand has been anantibody (Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218;Scott and Smith, 1990, Science 249:386-390). In many cases, the aim ofthe screening is to identify peptides from the library that mimic theepitopes to which the antibodies are directed. Thus, given an availableantibody, peptide libraries are excellent sources for identifyingepitopes or epitope-like molecules of that antibody (Yayon et al., 1993,Proc. Natl. Acad. Sci. USA 90:10643-10647).

While previous studies have succeeded in identifying epitopes andepitope-like molecules from peptide libraries, it has not been realizedin the prior art that this approach could be extended by using theidentified epitopes in a further round of screening of a peptide libraryto identify antibody-like molecules.

When it has been desired to obtain antibody-like molecules, the priorart has employed peptide libraries that contain naturally occurringantibody sequences. This has probably been due to the fact that specificbinding by antibodies is known to depend upon a complex structureinvolving various complementarity determining regions (CDRs), often fromboth heavy and light antibody chains. Short peptides would not beexpected to mimic such structures and longer peptides were thought to beunsuitable for display in the most commonly used libraries.

McCafferty et al., 1990, Nature 348:552-554 used PCR to amplifyimmunoglobulin variable (V) region genes and cloned those genes intophage expression vectors. The authors suggested that phage libraries ofV, diversity (D), and joining (J) regions could be screened withantigen. The phage that bound to antigen could then be mutated in theantigen-binding loops of the antibody genes and rescreened. The processcould be repeated several times, ultimately giving rise to phage whichbind the antigen strongly.

Marks et al., 1991, J. Mol. Biol. 222:581-597 also used PCR to amplifyimmunoglobulin variable (V) region genes and cloned those genes intophage expression vectors.

Kang et al., 1991, Proc. Natl. Acad. Sci. USA 88:4363-4366 created aphagemid vector that could be used to express the V and constant (C)regions of the heavy and light-chains of an antibody specific for anantigen. The heavy and light chain V-C regions were engineered tocombine in the periplasm to produce an antibody-like molecule with afunctional antigen binding site. Infection of cells harboring thisphagemid with helper phage resulted in the incorporation of theantibody-like molecule on the surface of phage that carried the phagemidDNA. This allowed for identification and enrichment of these phage byscreening with the antigen. It was suggested that the enriched phagecould be subject to mutation and further rounds of screening, leading tothe isolation of antibody-like molecules that were capable of evenstronger binding to the antigen.

Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133-4137 suggested thatnaive antibody genes might be cloned into phage display libraries. Thiswould be followed by random mutation of the cloned antibody genes togenerate high affinity variants.

In the prior art, peptide libraries have been screened with receptors toidentify receptor ligand-like peptides, but peptide libraries have notbeen considered useful for identifying such ligand-binding peptides asthose that mimic receptors.

Bass et al., 1990, Proteins: Struct. Func. Genet. 8:309-314 fused humangrowth hormone (hGH) to the carboxy terminus of the gene III protein ofphage fd. This fusion protein was built into a phagemid vector. Whencells carrying the phagemid were infected with a helper phage, about 10%of the phage particles produced displayed the fusion protein on theirsurfaces. These phage particles were enriched by screening with hGHreceptor-coated beads. It was suggested that this system could be usedto develop mutants of hGH with altered receptor binding characteristics.

Lowman et al., 1991, Biochemistry 30:10832-10838 used an improvedversion of the system of Bass et al. described above to select formutant hGH proteins with exceptionally high affinity for the hGHreceptor. The authors randomly mutagenized the hGH-pIII fusion proteinsat sites near the vicinity of 12 amino acids of hGH that had previouslybeen identified as being important in receptor binding.

Balass et al., 1993, Proc. Natl. Acad. Sci. USA 90:10638-10642 used aphage display library to isolate linear peptides that mimicked aconformationally dependent epitope of the nicotinic acetylcholinereceptor. This was done by screening the library with a monoclonalantibody specific for the conformationally dependent epitope. Themonoclonal antibody used was thought to be specific to the acetylcholinereceptor's binding site for its natural ligand, acetylcholine.

Citation or identification of any reference herein shall not beconstrued as an admission that such reference is available as prior artto the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to abtides. As used herein, the term"abtides" refers to peptides that mimic the binding specificity of alarger molecule such as an antibody or receptor. Abtides specificallybind to a ligand of interest, in which the ligand is a specific bindingpartner of the larger molecule (e.g. antibody or receptor). To identifythe abtides of the present invention, peptide libraries are screened ina two-step process. The first screening step uses an antibody (orantigen-binding derivative thereof) or receptor (or ligand-bindingderivative thereof) as a first target ligand. This step identifiespeptide sequences termed "epitopes" or "mimetopes" which specificallybind the first target ligand. In the case where an antibody orderivative thereof is used as the first target ligand, a mimetope willoften resemble, either functionally in terms of its binding capabilityand/or structurally in terms of its amino acid sequence, the epitoperecognized by the antibody used as the first ligand. An epitope ormimetope is then used as a second target ligand in a second screeningstep to identify a peptide sequence that specifically binds the epitopeor mimetope. Such peptides are known as "abtides." Surprisingly, it wasfound by the current inventor and is demonstrated herein that abtidespossess binding specificities strikingly similar to those possessed bythe first target ligands (usually antibodies or receptors) describedabove.

Abtides are useful since they mimic the binding specificities ofantibodies or receptors. Thus, they may be used in many instances whereantibodies or receptors may be used. The present invention furtherrelates to the use of abtides in the place of antibodies in assays suchas the many types of immunoassays known in the art. Abtides may take theplace of antibodies in such assays as, for example, enzyme-linkedimmunosorbent assays (ELISAs) or sandwich immunoassays. The inventionalso provides abtide compositions for use in therapy and diagnosis. In aspecific example, abtides have been discovered and demonstrated to beuseful in place of antibodies in enzyme-linked immunosorbent assays andin in vivo localization to prostate carcinoma in a xenograft model.

The use of abtides has many potential advantages over the use ofantibodies or receptors: the smaller size of abtides allows their easierproduction at lower cost, reduced immunogenicity, and may facilitatetheir in vivo delivery if such is desired; biological reactions andfunctions mediated by constant domains of antibodies, and cross-linkingof antibodies/receptors and resulting biological effects can be avoidedif desired.

4. BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be understood more fully by reference to thefollowing detailed description of the invention, examples of specificembodiments of the invention and the appended figures in which:

FIG. 1 shows in schematic diagram a general method for identifying anabtide by a two-step screening process. See Section 5.3 for a discussionof this method.

FIG. 2 shows the binding of biotinylated monoclonal antibody 7E11-C5 toimmobilized mimetope peptide 7E11-9.5. See Section 6.1.2.2 for details.

FIG. 3 shows similarities in the amino acid sequences of the CDR2L andCDR3L regions of monoclonal antibody 7E11-C5 and the 7E11-C5 abtides ofTable 2. The number of amino acids in the abtides that are similar tothe CDRs is indicated in parentheses, along with the percent homology.Dashes indicate gaps which have been added to improve the homology. Inthe case of clones 13 and 16, the homology with CDR2L was greatest ifthe sequence of CDR2L was reversed. The sequence shown for clone 14 isSEQ ID NO: 1; the sequence shown for clone 17 is SEQ ID NO: 2; Thesequence shown for clone 15 is SEQ ID NO: 3; the sequence shown forclone 13 is SEQ ID NO: 4; the sequence shown for clone 16 is SEQ ID NO:5; the sequence shown for CDR3L is SEQ ID NO: 6; the sequence shown forCDR2L is SEQ ID NO: 7; the sequence shown for CDR2L(rev) is SEQ ID NO:8.

FIG. 4 shows binding of abtides to the 7E11-9.5 mimetope peptide in adot blot assay as described in Section 6.1.2.1. Numbers along the leftside of the figure refer to the 7E11-C5 abtide that was spotted in theindicated position. The number 351 refers to the monoclonal antibody7E11-C5, used as a positive control. The numbers along the top of thefigure refer to the various dilutions of the abtide or the monoclonalantibody that were used.

FIG. 5 shows the binding of biotinylated mimetopes to immobilizedabtides. □ represents binding of mimetope peptideBiotin-LYANPGMYSRLHSPA-NH₂ (SEQ ID NO: 20) to 7E11-C5 abtide clone 14; ◯represents binding of mimetope peptide Biotin-LYANPGMYSRLHSPA-NH₂ (SEQID NO: 20) to 7E11-C5 abtide clone 17; ⋄ represents binding of mimetopepeptide Biotin-GMYSRLH-NH₂ (SEQ ID NO: 20) to 7E11-C5 abtide clone 14; Δrepresents binding of mimetope peptide Biotin-GMYSRLH-NH₂ (SEQ ID NO:20) to 7E11-C5 abtide clone 17. See Section 6.1.2.2 for details.

FIG. 6 shows the capture of an antigen from a lysate of LNCaP tumorcells by the monoclonal antibody 7E11-C5 and the 7E11-C5 abtide clone14. See Section 6.1.3 for details.

FIG. 7 shows the biodistribution of abtide clone 14-DPTA-¹¹¹ In in SCIDmice bearing human prostate carcinoma LNCaP xenograft tumors 2 hours (,bar on the left for each pair of bars) or 4 hours (, bar on the rightfor each pair of bars) post-injection of 2 μg of peptide, specificactivity 32 μCi/μg. See Section 6.1.4 for details.

FIG. 8 shows the biodistribution of abtide clone 17-DPTA-¹¹¹ -In in fourSCID mice bearing human prostate LNCaP carcinoma xenograft tumors 2hours (, leftmost bar for each group of four bars, mouse 1; , second barfrom left for each group of four bars, mouse 6) or five hours (, thirdbar from left for each group of four bars, mouse 2; , rightmost bar foreach group of four bars, mouse 4) post-injection of 0.02 μg of peptide,specific activity 2.4 μCi/ng. See Section 6.1.4 for details.

FIG. 9 shows the biodistribution of ¹¹¹ -In labeled control irrelevantpeptide in SCID mice bearing human prostate carcinoma LNCaP xenografttumors 2 hours ( leftmost bar for each group of five bars; second barfrom left for each group of five bars) or 5 hours ( third bar from leftfor each group of five bars; fourth bar from left for each group of fivebars; rightmost bar for each group of five bars) post-injection of 1.5μg of peptide, specific activity 30 μCi/μg. See Section 6.1.4 fordetails.

FIG. 10 schematically illustrates the construction of the R26 TSARlibrary. The R26 expression library was constructed essentially asdescribed for the TSAR-9 library that is described in PCT publication WO94/18318, dated Aug. 18, 1994, except for the modifications depicted inFIG. 10. The oligonucleotide assembly process depicted in FIG. 10results in expression of peptides with the following amino acidsequence:

    S(S/R)X.sub.12 πAδX.sub.12 SR                     (SEQ ID NO: 25),

where

π=S, P, T or A; and

δ=V, A, D, E or G. ctgtgcctcgagB(NNB)₁₂ Nccgcgg is SEQ ID NO: 87;ctgtgctctaga(VNN)₁₂ VNccgcgg is SEQ ID NO: 88; tcgagB(NNB)₁₂ Nccgcgg isSEQ ID NO: 89; ctagt(VNN)₁₂ VNccgcgg is SEQ ID NO: 90; SHSS(S/R)x₁₂πAδX₁₂ SRPSRT is SEQ ID NO: 91.

FIG. 11 schematically illustrates the construction of the D38 TSARlibrary. The D38 expression library was constructed essentially asdescribed for the TSAR-9 library that is described in PCT publication WO94/18318, dated Aug. 18, 1994, except for the modifications depicted inFIG. 11. GTGTGTCTCGAGN(NNB)₂₀ NACGCCAN is SEQ ID NO: 92;GTTGTGTCTAGA(VANN)₁₅ VNTGGCGTN is SEQ ID NO: 93; TCGAGN(NNB)₂₀ NACGCCANis SEQ ID NO: 94; CTAGA(VNN)₁₅ VNTGGCGTN is SEQ ID NO: 95; HSS(S/R)X₂₀(Y/H/N/D)A(I/M/T/N/K/S/R)X₁₅ SR is SEQ ID NO: 96.

FIG. 12 schematically illustrates the construction of the DC43 TSARlibrary. The DC43 expression library was constructed essentially asdescribed for the TSAR-9 library that is described in PCT publication WO94/18318, dated Aug. 18, 1994, except for the modifications depicted inFIG. 12. GTGTGTCTCGAGN(NNB)₂₀ GGTTGTGGT is SEQ ID NO: 97;GTTGTGTCTAGA(VNN)₂₀ ACCACAACC is SEQ ID NO: 98; TCGAGN(NNB)₂₀ GGTTGTGGTis SEQ ID NO: 99; CTAGA(VNN)₂₀ ACCACAACC is SEQ ID NO: 100; HSS(S/R)X₂₀GCGX₂₀ SR is SEQ ID NO: 101.

FIG. 13 schematically illustrates the oligonucleotides used to constructthe polymorphic epithelial mucin (PEM) abtide saturation mutagenesisTSAR library (See Section 6.2.2).GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP is SEQ ID NO: 51;ggsgcsccsgtstgsagsggsaasccscgstgsagsggsccsggsggsttsaastgsccsGGCTGCGGG isSEQ ID NO: 102;sggsccsttsttscgsgasccsccscgsgcsggsgtsaasgtsttscasatsggsccsttCCCGCAGCC isSEQ ID NO: 103.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to abtides.

As used herein, the term "abtides" refers to peptides that mimic thebinding specificity of a larger molecule such as an antibody orreceptor. Abtides specifically bind to a ligand of interest, in whichthe ligand is a specific binding partner of the mimicked larger molecule(e.g. antibody or receptor). To identify the abtides of the presentinvention, peptide libraries are typically screened in a two-stepprocess (see FIG. 1). The first screening step uses an antibody (orantigen-binding derivative thereof) or receptor (or ligand-bindingderivative thereof) as a first target ligand. This step identifiespeptide sequences termed "epitopes" or "mimetopes" which specificallybind the first target ligand. If the first screening step uses anantibody and the peptide identified contains the amino acid sequence ofthe natural antigen that is responsible for the specific binding of theantigen to the antibody, then the identified peptide is said to be anepitope; if the identified peptide does not contain the sequence of thenatural antigen, then the identified peptide is said to be a mimetope.In the case where an antibody or derivative thereof is used as the firsttarget ligand, a mimetope will often resemble, either functionally interms of its binding capability and/or structurally in terms of itsamino acid sequence, the epitope recognized by the antibody used as thefirst ligand.

A mimetope is then used as a second target ligand in a second screeningstep to identify a peptide sequence that specifically binds the epitopeor mimetope. Such peptides are known as "abtides." Abtides possessbinding specificities similar to those possessed by the first targetligands (usually antibodies or receptors) described above.

In a specific embodiment, the antibody or derivative thereof used in thefirst screening step recognizes a tumor antigen, preferably a humantumor antigen, most preferably of a malignant tumor.

The present invention provides a method to successfully screen againstvery small peptide or protein targets, e.g. 5 to 40 amino acids,preferably 10 to 20 amino acids. To date, screening against such targetshas not been successful. The methods of the present invention increasethe likelihood that the abtide obtained will bind its target in acomplex or structurally dependent fashion.

Abtides are useful since they mimic the binding specificities ofantibodies or receptors. Thus, they may be used in many instances whereantibodies or receptors may be used. The present invention furtherrelates to the use of abtides in the place of antibodies in assays suchas the many types of immunoassays known in the art. Abtides may take theplace of antibodies in such assays as, for example, enzyme-linkedimmunosorbent assays (ELISAs) or sandwich immunoassays. The inventionalso provides abtide compositions for use in therapy and diagnosis ofdisease. In a specific example, abtides have been produced anddemonstrated to be useful in place of antibodies in enzyme-linkedimmunosorbent assays and in in vivo localization to prostate carcinomain xenograft models.

The use of abtides has many potential advantages over the use ofantibodies or receptors: the smaller size of abtides allows their easierproduction at lower cost, reduced immunogenicity, and facilitates theirin vivo delivery if such is desired; biological reactions and functionsmediated by constant domains of antibodies, and cross-linking ofantibodies/receptors and resulting biological effects can be avoided ifdesired.

5.1. Peptide Libraries for Use in Identifying Abtides

The abtides of the present invention can be identified from a chemicallysynthesized peptide library or a biologically expressed peptide library.If a biological peptide expression library is used, the nucleic acidwhich encodes the peptide which binds to the ligand of choice can berecovered, and then sequenced to determine its nucleotide sequence andhence deduce the amino acid sequence that mediates binding.Alternatively, the amino acid sequence of an appropriate binding domaincan be determined by direct determination of the amino acid sequence ofa peptide selected from a peptide library containing chemicallysynthesized peptides. In a less preferred aspect, direct amino acidsequencing of a binding peptide selected from a biological peptideexpression library can also be performed.

In a preferred embodiment of the present invention, the abtides areadvantageously identified from random peptide libraries. Typically,random peptide libraries will be encoded by synthetic oligonucleotideswith a plurality of variant nucleotide positions having the potential toencode all 20 naturally occurring amino acids. The sequence of aminoacids encoded by the variant nucleotides is unpredictable andsubstantially random. The terms "unpredicted", "unpredictable" and"substantially random" are used interchangeably with respect to theamino acids encoded and are intended to mean that the variantnucleotides at any given position are such that it cannot be predictedwhich of the 20 naturally occurring amino acids will appear at thatposition. These variant nucleotides are the product of random chemicalsynthesis. The biological random peptide libraries envisioned for useinclude those in which a bias has been introduced into the randomsequence, e.g., to disfavor stop codon usage.

5.1.1. Chemically Synthesized Peptide Libraries

The peptide libraries used in the present invention may be librariesthat are chemically synthesized in vitro. Examples of such libraries aregiven in Fodor et al., 1991, Science 251:767-773, which describes thesynthesis of a known array of short peptides on an individualmicroscopic slide; Houghten et al., 1991, Nature 354:84-86, whichdescribes mixtures of free hexapeptides in which the first and secondresidues in each peptide were individually and specifically defined. Lamet al., 1991, Nature 354:82-84, which describes a split synthesisscheme; Medynski, 1994, Bio/Technology 12:709-710, describes splitsynthesis and T-bag synthesis methods as well. See also Gallop et al.,1994, J. Medicinal Chemistry 37:1233-1251.

PCT publication WO 91/05058, dated Apr. 18, 1991, is directed to randomlibraries containing semi-random nucleotide sequences. The semi-randomnucleotide sequences are transcribed in vitro under conditions such thatpolysomes are produced. The polysomes are screened for binding to asubstance of interest. Those polysomes that bind to the substance ofinterest are recovered. The RNA from those polysomes is used toconstruct cDNA, which is expressed to produce polypeptides.

Screening to identify peptides which bind to a ligand of choice can becarried out by methods well known in the art.

In a specific embodiment, the total number of unpredictable amino acidsin the peptides of the chemical library used for screening is greaterthan or equal to 5 and less than or equal to 25; in other embodimentsthe total is in the range of 5-15 or 5-10 amino acids, preferablycontiguous amino acids.

While a binding domain can be identified from chemically synthesizedpeptide libraries, such a domain would be small (i.e. less than 10 aminoacids, and most probably 5-6 amino acids, in length). Therefore, use ofa chemically synthesized peptide library is less preferred for thesecond screening step involved in isolating abtides than is use in thesecond screening step of biological peptide libraries containingunpredictable sequences of greater length, described below.

5.1.2. Biological Peptide Libraries

In another embodiment, biological peptide libraries are used to identifyabtides. Many suitable biological peptide libraries are known in the artand can be used.

According to this second approach, involving recombinant DNA techniques,peptides have been expressed in biological systems as either solublefusion proteins or viral capsid fusion proteins:

A number of peptide libraries according to this approach have used theM13 phage. Although the N-terminus of the viral capsid protein, proteinIII (pIII), has been shown to be necessary for viral infection, theextreme N-terminus of the mature protein does tolerate alterations suchas insertions. Accordingly, various peptide libraries, in which thediverse peptides are expressed as pIII fusion proteins, are known in theart; these libraries can be used to identify abtides. Examples of suchlibraries are described below.

Scott and Smith, 1990, Science 249:386-390 describe construction andexpression of an "epitope library" of hexapeptides on the surface ofM13. The library was made by inserting a 33 base pair Bgl-I digestedoligonucleotide sequence into an Sfi I digested phage fd-tet, i.e.,fUSE5 RF. The 33 base pair fragment contains a random or "degenerate"coding sequence (NNK)₆ where N represents G, A, T or C and K representsG or T. The authors stated that the library consisted of 2×10⁸recombinants expressing 4×10⁷ different hexapeptides; theoretically,this library expressed 69% of the 6.4×10⁷ possible peptides (20⁶).Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382 alsodescribed a somewhat similar library of hexapeptides expressed as pIIIgene fusions of M13 fd phage. PCT publication WO 91/19818 dated Dec. 26,1991 by Dower and Cwirla describes a similar library of pentameric tooctameric random amino acid sequences.

Devlin et al., 1990, Science, 249:404-406, describes a peptide libraryof about 15 residues generated using an (NNS) coding scheme foroligonucleotide synthesis in which S is G or C.

Christian and colleagues have described a phage display library,expressing decapeptides (Christian et al., 1992, J. Mol. Biol.227:711-718). The starting DNA was generated by means of anoligonucleotide comprising the degenerate codons [NN(G/T)]₁₀ with aself-complementary 3' terminus. This sequence, in forming a hairpin,creates a self-priming replication site which could be used by T4 DNApolymerase to generate the complementary strand. The double-stranded DNAwas cleaved at the SfiI sites at the 5' terminus and hairpin for cloninginto the fUSE5 vector described by Scott and Smith, supra.

Lenstra, 1992, J. Immunol. Meth. 152:149-157 describes construction of alibrary by a laborious process encompassing annealing oligonucleotidesof about 17 or 23 degenerate bases with an 8 nucleotide long palindromicsequence at their 3' ends. This resulted in the expression of randomhexa- or octa-peptides as fusion proteins with the β-galactosidaseprotein in a bacterial expression vector. The DNA was then convertedinto a double-stranded form with Klenow DNA polymerase, blunt-endligated into a vector, and then released as Hind III fragments. Thesefragments were then cloned into an expression vector at the sequenceencoding the C-terminus of a truncated β-galactosidase to generate 10⁷recombinants.

Other biological peptide libraries which can be used include thosedescribed in U.S. Pat. No. 5,270,170 dated Dec. 14, 1993 and PCTPublication No. WO 91/19818 dated Dec. 26, 1991. Also suitable are thosein U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,198,346, and U.S. Pat. No.5,223,409, all to Ladner et al.

The biological peptide libraries discussed above are meant to beillustrative and not limiting. It will be recognized by one of skill inthe art that many other biological peptide libraries disclosed invarious publications may be suitable for use in the practice of thepresent invention.

The protein pVIII is a major M13 viral capsid protein, which can alsoserve as a site for expressing peptides on the surface of M13 viralparticles, in the construction of random peptide libraries.

While it would be understood by one skilled in the art that as few as 5amino acids can constitute a binding domain, the average functionaldomain within a natural protein is considered to be about 40 aminoacids. Thus, the random peptide libraries from which the abtides of thepresent invention are preferably identified encode peptides having inthe range of 5 to 200 total variant amino acids. Although it iscontemplated that biologically expressed random peptide librariesdisplaying short random inserts (i.e. less than 20 amino acids inlength) could be used to identify abtides of the invention, the mostpreferred biologically expressed random peptide libraries for use in theinvention are those in which the displayed peptide has 20 or greaterunpredictable amino acids i.e. preferably in the range of 20 to 100, andmost preferably 20 to 50 amino acids, as exemplified by the TSARlibraries described in PCT publication WO 91/12328, dated Aug. 22, 1991,and PCT publication WO 94/18318, dated Aug. 18, 1994.

To identify abtides, particularly in the second screening step, theinvention preferably uses libraries of greater complexity than arecommonly employed in the art. The conventional teaching in the randompeptide library art is that the length of inserted oligonucleotidesshould be kept short, encoding preferably fewer than 15 and mostpreferably about 6-8 amino acids. However, not only can librariesencoding more than about 20 amino acids be constructed, but suchlibraries can be advantageously screened to identify peptides havingbinding specificity for a variety of ligands. Such libraries with longerlength inserts are exemplified by the TSAR libraries, described in PCTpublication WO 91/12328, dated Aug. 22, 1991, and PCT publication WO94/18318, dated Aug. 18, 1994.

These PCT publications disclose that the use of libraries composed oflonger length oligonucleotides has many advantages.

Libraries composed of longer length oligonucleotides afford the abilityto identify peptides in which a short sequence of amino acids is commonto or shared by a number of peptides binding a given ligand, i.e.,library members having shared binding motifs. The use of longer lengthlibraries also affords the ability to identify peptides which do nothave any shared sequences with other peptides but which neverthelesshave binding specificity for the same ligand.

When screened by the method of the present invention, libraries havinglarge inserted oligonucleotide sequences provide the opportunity toidentify or map binding sites which encompass not only a few contiguousamino acid residues, i.e., simple binding sites, but also those whichencompass discontinuous amino acids, i.e., complex binding sites, andmay afford the complex binding characteristic of antibodies andreceptor-like molecules.

Additionally, the large size of the inserted synthesizedoligonucleotides of certain libraries provides the opportunity for thedevelopment of secondary and/or tertiary structure in the potentialbinding peptides and in sequences flanking the actual binding site inthe binding domain. Secondary and tertiary structure often significantlyaffect the ability of a sequence to mediate binding, as well as thestrength and specificity of any binding which occurs. Such complexstructural effects are not possible when only small lengtholigonucleotides are used in libraries. It may be that secondary andtertiary structures are especially important in the identification ofabtides since abtides mimic the binding of large molecules such asantibodies. It is well known that the antigen binding properties ofantibodies depend in most instances upon several different regions ofthe heavy chain (complementarity determining regions) and upon regionscontributed by the light chain as well.

Therefore, it is contemplated that the most preferred binding domainsfor identifying the abtides of the present invention will be those frombiologically expressed random peptide libraries in which the displayedpeptide is 20 or greater amino acids in length. Examples of such randompeptide libraries are the TSAR libraries, described in PCT publicationWO 91/12328, dated Aug. 22, 1991, and PCT publication WO 94/18318, datedAug. 18, 1994.

In one embodiment, the library utilized in the present invention is alinear, non-constrained library. As would be understood by one in theart having considered the present disclosure, in another specificembodiment, "constrained", "structured" or "semi-rigid" random peptidelibraries could also be used in the present methods to identify abtides.Typically, these libraries express peptides that are substantiallyrandom but contain a small percentage of fixed residues within orflanking the random sequences that have the result of conferringstructure or some degree of conformational rigidity to the peptide. In asemirigid peptide library, the plurality of synthetic oligonucleotidesexpress peptides that are each able to adopt only one or a small numberof different conformations that are constrained by the positioning ofcodons encoding certain structure conferring amino acids in or flankingthe synthesized variant or unpredicted oligonucleotides. Unlike linear,unconstrained libraries in which the plurality of proteins expressedpotentially adopt thousands of short-lived different conformations, in asemirigid peptide library, the plurality of proteins expressed can adoptonly a single or a small number of conformations. Such libraries areexemplified by the TSAR-13 and TSAR-14 libraries described in PCTpublication WO 94/18318, dated Aug. 18, 1994; by a library of random 6amino acid sequences, each flanked by invariant cysteine residues(O'Neil et al., 1992, Proteins 14:509-515); and by those librariesdisclosed in PCT Publication No. WO 94/11496, dated May 26, 1994 (Huse).

In a preferred embodiment, a biological peptide library that is a randompeptide "TSAR" library is screened to identify an abtide. TSARs is anacronym for "Totally Synthetic Affinity Reagents" as described in PCTpublication WO 91/12328, dated Aug. 22, 1991, and PCT publication WO94/18318, dated Aug. 18, 1994. TSAR libraries, their construction anduse, and specific examples of TSAR libraries, are described in detail inthose PCT publications. Nucleic acids encoding TSARs or a TSAR portionwhich mediates binding to the ligand used for screening can be used toidentify the abtides of the present invention.

A TSAR may be a heterofunctional fusion protein, said fusion proteincomprising (a) a binding domain encoded by an oligonucleotide comprisingunpredictable nucleotides in which the unpredictable nucleotides arearranged in one or more contiguous sequences, wherein the total numberof unpredictable nucleotides is greater than or equal to about 60 andless than or equal to about 600, and optionally, (b) an effector domainencoded by an oligonucleotide sequence which is a protein or peptidethat enhances expression or detection of the binding domain.

Alternatively, a TSAR may be a heterofunctional fusion protein asdescribed above but in which the contiguous sequences are flanked byinvariant residues designed to encode amino acids that confer a desiredstructure to the binding domain of the expressed heterofunctional fusionprotein.

In addition to TSAR libraries, other libraries for use in the presentinvention may be those wherein the library is a library of recombinantvectors that express a plurality of heterofunctional fusion proteins,said fusion proteins comprising a binding domain encoded by anoligonucleotide comprising unpredictable nucleotides in which theunpredictable nucleotides are arranged in one or more contiguoussequences, wherein the total number of unpredictable nucleotides isgreater than or equal to about 15 and less than or equal to about 600.

5.2. Abtides

An abtide is typically a peptide that mimics, with respect to bindingspecificity, and possibly other characteristics (e.g., binding affinity,sequence, etc.) a large molecule such as an antibody or receptor.However, an abtide is generally much smaller than an antibody orreceptor. An abtide is generally a peptide of about 5 to 200 aminoacids. Preferably, an abtide is a peptide of about 10 to 100 aminoacids. Most preferably, an abtide is a peptide of about 20 to 50 aminoacids. In addition to the amino acid sequences which are responsible forthe abtide's specific binding properties, an abtide may be linked toadditional amino acid sequences or additional non-amino acid sequences.Such additional sequences may aid in the identification or isolation ofthe abtide. Or, they may aid in the biodistribution, stability, ordiagnostic or therapeutic effectiveness of the abtide when the abtide isused diagnostically or therapeutically.

The abtides may be linked to a variety of non-peptide moieties. Suchmoieties might include toxins; drugs; polysaccharides; nucleotides;oligonucleotides; labels such as radioactive substances (e.g. ¹¹¹ In,¹²⁵ I, ¹³¹ I, ^(99m) Tc, ²¹² B, ⁹⁰ Y, ¹⁸⁶ Rh); biotin; fluorescent tags;imaging reagents (e.g. those described in U.S. Pat. No. 4,741,900 andU.S. Pat. No. 5,326,856); hydrocarbon linkers (e.g., an alkyl group orderivative thereof) conjugated to a moiety providing for attachment to asolid substratum, or to a moiety providing for easy separation (e.g., ahapten recognized by an antibody bound to a magnetic bead), etc. Linkageof the peptide to the non-peptide moiety may be by any of severalwell-known methods in the art.

In addition, in an embodiment in which an abtide has a free amino- orcarboxy-terminus, such termini can be modified by known methods, e.g.,to provide greater resistance to degradation, greater cell permeability,greater solubility, etc., e.g., by acetylation, biotinylation, fattyacylation, etc. at the amino-terminus; amidation at thecarboxy-terminus; or the abtide can be stabilized by inclusion of Damino acids, nonnatural amino acids or glycosyl amino acids, etc.

The abtides of the invention are preferably made by commonly knownmethods of chemical synthesis, e.g., as described by way of example inSection 5.6 and its subsections.

Alternatively, abtides (or portions thereof) can be obtained byrecombinant expression of a nucleic acid encoding the desired abtide inan appropriate host, by well-known methods.

Occasionally, it may happen that not all of the amino acids in theidentified peptide will be necessary for the binding function of anabtide. Where it is desired to decrease the size of the abtide, methodscan be used to identify portions of the determined synthetic amino acidor nucleotide sequences which respectively mediate binding, or encodethe sequences which mediate binding, as described in Section 5.4 below.

5.3. Screening of Peptide Libraries to Identify Abtides

The process of identifying abtides from a peptide library comprises twodistinct screening steps. The first step is designed to identifyepitopes or ligands with binding specificity for the larger molecule ofinterest, e.g. antigen mimics, receptor-ligand mimics, and the like. Inthat first step, a peptide library is screened with a ligand thatpossesses a specific, often complex, binding site of interest. Thosepeptides in the library that are specific binding partners of the ligandbind to the ligand and are readily recoverable because of this specificbinding. An example of a ligand suitable for use in this first screeningstep would be an antibody, inherently possessing an antigen bindingsite, or an antigen-binding derivative thereof. Another example would bea receptor e.g. the epidermal growth factor receptor or the plateletderived growth factor receptor. These particular receptors possessspecific binding sites for epidermal growth factor and platelet derivedgrowth factor, respectively. Other receptors are known in the art andare also potential ligands, for example, the estrogen receptor, thevarious acetylcholine receptors, the human growth hormone receptor, etc.

Molecules comprising those peptide sequences in the library that areidentified by the first screening step will be referred to herein asepitopes or mimetopes. For example, in the case where the first ligandis an antibody, the peptides in the library that are identified asspecific binding partners of the antibody would be known as antigenepitopes or mimetopes.

The peptides that are isolated in the first screening step are thenpreferably analyzed, as, for example, by DNA sequencing of the bindingdomain of the phage that encode the peptides if the library used was aphage library. The DNA sequence of the binding domain encodes the aminoacid sequence of the epitope or mimetope peptide. Due to the knownrelationship between DNA sequences and their encoded amino acidsequences, obtaining the DNA sequence of the epitope or mimetope allowsthe determination of the amino acid sequence of the epitope or mimetope.Alternatively, if the library used was a chemical library, direct aminoacid sequencing of the peptide epitope or mimetope can be carried out bywell known methods in the art.

In a specific embodiment, sequences of different peptide mimetopesidentified in the first screening step can be compared to determine aconsensus mimetope sequence.

Once the amino acid sequence of the epitope or mimetope is known (or aportion thereof, which mediates binding), a molecule, preferably apeptide, is produced comprising that amino acid sequence which mediatesbinding. This peptide may be synthesized chemically, or, alternatively,may be produced by methods involving recombinant DNA. This peptide maycontain only the amino acids of the epitope or mimetope or, preferably,it may contain additional amino acids or non-amino acid moieties to aidin identifying or recovering the epitope or mimetope peptide and any newpeptide binders found in the subsequent screening step discussed below.

In the second screening step, the epitope or mimetope that wasidentified in the first screening step is used as a ligand for thesecond screening step. The second screening step identifies peptideswith binding specificity for the epitope or mimetope and thatsurprisingly mimic the binding specificity of the antibody or receptorthat was used as ligand in the first screening step. In other words, thesecond screening step yields peptides with antibody or receptor-likebinding activity for antigens or receptor ligands that are known asabtides.

FIG. 1 is a schematic representation of an exemplary two-step screeningprocess used to identify abtides.

In a particular embodiment of the invention, it may be that the epitopeto which an antibody specifically binds is known. For example, themonoclonal antibody SM-3 that specifically binds the polymorphicepithelial mucin (PEM) found on human breast carcinoma cells has beenshown to be specific for the epitope defined by the amino acid sequenceVTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 9) (Burchell et al., 1989, Int.J. Cancer 44:691-696). In such cases, the first screening step describedabove may be dispensed with. A peptide comprising the sequence of theepitope for which the antibody is specific can be synthesized and usedin the second screening step described above in order to identifyabtides of the antibody.

It may also be that the portion of a "receptor-ligand" (i.e., a ligandwhich specifically binds to a receptor) to which a receptor specificallybinds is known. For example, it has been shown thatgranulocyte/macrophage colony stimulating factor (GM-CSF) binds to theGM-CSF receptor through amino acids 88-121(HCPPTPETSCATQTITFESFKENLKDFLLVIPFDC [SEQ ID NO: 22]) of GM-CSF. Itshould be possible to synthesize a peptide corresponding to the portionof the receptor-ligand that has been shown to be responsible forspecific binding to the receptor and to use such a peptide in the secondscreening step of the methods of the present invention in order toidentify an abtide of the receptor.

In an embodiment, a molecule comprises a peptide or a binding portionthereof which binds to a ligand of interest, which peptide is identifiedby a method comprising: screening a random peptide library with a ligandof interest, said ligand of interest being a peptide having a length ofbetween 5 and 40 amino acids, to identify a peptide that specificallybinds to the ligand of interest, in which the ligand of interest is alsospecifically bound by an antibody or a receptor.

In another embodiment, a molecule comprises a peptide which binds to asubstance of interest, which peptide is identified by a methodcomprising: screening a random peptide library with a ligand, saidligand being a peptide of 36 amino acids or fewer, in which the ligandis an epitope of an antigen that is specifically bound by an antibody orin which the ligand represents the portion of a receptor-ligand that isresponsible for the specific binding of the receptor to thereceptor-ligand.

As used in the present invention, a ligand is a substance for which itis desired to isolate a specific binding partner from a peptide library.A ligand can function as a lock, i.e., a large polypeptide or proteinanalogous to a lock into which a smaller specific binding partner fitsas a key; or a ligand can function as a key which fits into andspecifically binds a larger binding partner or lock.

In this invention, an epitope or mimetope is typically a peptide thatacts as a key; it is identified by screening a peptide library forpeptides that fit into and bind the specific binding site of a largermolecule which acts as a lock, e.g. antibody or receptor. If the largermolecule is an antibody and the peptide identified contains the portionof the amino acid sequence of the natural antigen that is responsiblefor the specific binding of the antigen to the antibody, then theidentified peptide is said to be an epitope; if the identified peptidedoes not contain the sequence of the natural antigen, then theidentified peptide is said to be a mimetope.

In a specific embodiment, a mimetope is identified by screening apeptide library with an antibody or antibody fragment. Mimetopes thusidentified functionally mimic the antigen to which the antibody binds inthat the mimetopes also specifically bind with the antibody. In somecases, if the antigen is a protein or polypeptide, the mimetopes mayshare amino acid sequence motifs with the antigen. In anotherembodiment, a mimetope is identified by screening a peptide library witha receptor or receptor fragment. Mimetopes thus identified functionallymimic the natural ligand of the receptor.

The peptide libraries that are used in the first and second screeningsteps may be the same or different. In one embodiment, a peptide librarycontaining small random inserts (from about 6 to about 15 amino acids)is used in the first screening step.

In the second screening step, it may be desirable to use a largerpeptide library. Such larger libraries preferably express peptides ofabout 20 to 200 random amino acids. Examples of such larger librariesare the TSAR libraries described in PCT publication WO 91/12328, datedAug. 22, 1991, and in PCT Publication WO 94/18318, dated Aug. 18, 1994.

Biological or chemically synthesized peptide libraries can be used ineither the first or second screenings. The peptide libraries used in thepresent invention may have a plurality of residues that are random, i.e.residues for which the amino acid occupying that residue cannot bepredicted in advance. Such libraries are said to be random peptidelibraries.

A preferred method for identifying abtides comprises screening a libraryof recombinant vectors that express a plurality of heterofunctionalfusion proteins, said fusion proteins comprising (a) a binding domainencoded by an oligonucleotide comprising unpredictable nucleotides inwhich the unpredictable nucleotides are arranged in one or morecontiguous sequences, wherein the total number of unpredictablenucleotides is greater than or equal to about 15 and less than or equalto about 600, and optionally, (b) an effector domain encoded by anoligonucleotide sequence which is a protein or peptide that enhancesexpression or detection of the binding domain. Screening is done bycontacting the plurality of heterofunctional fusion proteins with aligand under conditions conducive to ligand binding and then isolatingthe fusion proteins which bind to the ligand. The methods of theinvention further preferably comprise determining the nucleotidesequence encoding the binding domain of the heterofunctional fusionprotein identified to determine the DNA sequence that encodes thebinding domain and simultaneously to deduce the amino acid sequence ofthe mimetope used in the second screen. Nucleotide sequence analysis canbe carried out by any method known in the art, including but not limitedto the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560),the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl, Acad. Sci.U.S.A. 74:5463), the use of SEQUENASE™ T7 DNA polymerase (Tabor andRichardson, U.S. Pat. No. 4,795,699; U.S. Biochemical Corp.), or TAQ™polymerase, or use of an automated DNA sequenator (e.g., AppliedBiosystems, Foster City, Calif.).

Alternatively, the libraries used to screen for mimetopes and abtides ofthe invention have unpredictable nucleotides arranged in one or morecontiguous sequences that are flanked by invariant residues designed toencode amino acids that confer a desired structure to the binding domainof the expressed heterofunctional fusion protein.

Once a suitable peptide library has been constructed (or otherwiseobtained), the library is screened to identify peptides having bindingaffinity for a ligand of choice. Screening the libraries can beaccomplished by any of a variety of methods known to those of skill inthe art. See, e.g., the following references, which disclose screeningof peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol.251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al.,1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl.Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt etal., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellingtonet al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No.5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; and Rebarand Pabo, 1993, Science 263:671-673. See also PCT publication WO94/18318, dated Aug. 18, 1994.

If the libraries are expressed as fusion proteins with a cell surfacemolecule, then screening is advantageously achieved by contacting thevectors with an immobilized target ligand and harvesting those vectorsthat bind to said ligand. Such useful screening methods, designated"panning" techniques are described in Fowlkes et al., 1992,BioTechniques 13:422-427. In panning methods useful to screen thelibraries, the target ligand can be immobilized on plates, beads, suchas magnetic beads, sepharose, etc., or on beads used in columns. Inparticular embodiments, the immobilized target ligand can be "tagged",e.g., using such as biotin, 2-fluorochrome, e.g. for FACS sorting.

In one embodiment, presented by way of example but not limitation,screening a library of phage expressing random peptides on phage andphagemid vectors can be achieved by using magnetic beads as described inPCT publication WO 94/18318, dated Aug. 18, 1994.

Alternatively, as yet another non-limiting example, screening a libraryof phage expressing random peptides can be achieved by panning usingmicrotiter plates. In a preferred method for recovering the phage boundto the wells of the microtiter plates, a pH change is used.

By way of another example, the libraries expressing random peptides as asurface protein of either a vector or a host cell, e.g., phage orbacterial cell, can be screened by passing a solution of the libraryover a column of a ligand immobilized to a solid matrix, such assepharose, silica, etc., and recovering those phage that bind to thecolumn after extensive washing and elution.

By way of yet another example, weak binding library members can beisolated based on retarded chromatographic properties. According to onemode of this embodiment for screening, fractions are collected as theycome off the column, saving the trailing fractions (i.e., those membersthat are retarded in mobility relative to the peak fraction are saved).These members are then concentrated and passed over the column a secondtime, again saving the retarded fractions. Through successive rounds ofchromatography, it is possible to isolate those that have some affinity,albeit weak, to the immobilized ligand. These library members areretarded in their mobility because of the millions of possible ligandinteractions as the member passes down the column. In addition, thismethodology selects those members that have modest affinity to thetarget, and which also have a rapid dissociation time.

If desired, the oligonucleotides encoding the binding domain selected inthis manner can be mutagenized, expressed and rechromatographed (orscreened by another method) to discover improved binding activity. Inparticular, saturation mutagenesis can be carried out using syntheticoligonucleotides synthesized from "doped" nucleotide reservoirs. Thedoping is carried out such that the original peptide sequence isrepresented only once in 10⁶ unique clones of the mutagenizedoligonucleotide. The assembled oligonucleotides are cloned into aparental TSAR vector. Preferably, the vector is m663 (Fowlkes et al.,1992, BioTechniques 13:422-427). m663 is able to make blue plaques whengrown in E. coli stain JM101 or DH5αF'. A library of greater than 10⁶ ispreferred; however a library of 10⁵ is sufficient to isolate TSAR phagedisplaying peptide domains with increased selectivity for binding to thetarget ligand.

According to another alternative method, screening a library of can beachieved using a method comprising an "enrichment" step and a filterlift step as follows.

Random peptides from an expressed library capable of binding to a givenligand ("positives") are initially enriched by one or two cycles ofpanning or affinity chromatography, as described above. The goal is toenrich the positives to a frequency of about >1/10⁵. Followingenrichment, a filter lift assay is conducted. For example, approximately1-2×10⁵ phage, enriched for binders, are added to 500 μl of log phase E.coli and plated on a large LB-agarose plate with 0.7% agarose in broth.The agarose is allowed to solidify, and a nitrocellulose filter (e.g.,0.45μ) is placed on the agarose surface. A series of registration marksis made with a sterile needle to allow re-alignment of the filter andplate following development as described below. Phage plaques areallowed to develop by overnight incubation at 37° C. (the presence ofthe filter does not inhibit this process). The filter is then removedfrom the plate with phage from each individual plaque adhered in situ.The filter is then exposed to a solution of BSA or other blocking agentfor 1-2 hours to prevent non-specific binding of the ligand (or"probe").

The probe itself is labeled, for example, either by biotinylation (usingcommercial NHS-biotin) or direct enzyme labeling, e.g., with horseradish peroxidase (HRP) or alkaline phosphatase. Probes labeled in thismanner are indefinitely stable and can be re-used several times. Theblocked filter is exposed to a solution of probe for several hours toallow the probe to bind in situ to any phage on the filter displaying apeptide with significant affinity to the probe. The filter is thenwashed to remove unbound probe, and then developed by exposure to enzymesubstrate solution (in the case of directly labeled probe) or furtherexposed to a solution of enzyme-labeled avidin (in the case ofbiotinylated probe). In a preferred method, an HRP-labeled probe isdetected by ECL western blotting methods (Amersham, Arlington Heights,Ill.), which involves using luminol in the presence of phenol to yieldenhanced chemiluminescence detectable by brief exposure of film byautoradiography, in which the exposed areas of film correspond topositive plaques on the original plate. Where an enzyme substrate isused, positive phage plaques are identified by localized deposition ofcolored enzymatic cleavage product on the filter which corresponds toplaques on the original plate. The developed filter or film, as the casemay be, is simply realigned with the plate using the registration marks,and the "positive" plaques are cored from the agarose to recover thephage. Because of the high density of plaques on the original plate, itis usually impossible to isolate a single plaque from the plate on thefirst pass. Accordingly, phage recovered from the initial core arere-plated at low density and the process is repeated to allow isolationof individual plaques and hence single clones of phage.

Successful screening experiments are optimally conducted using 3 roundsof serial screening. The recovered cells are then plated at a lowdensity to yield isolated colonies for individual analysis. Theindividual colonies are selected and used to inoculate LB culture mediumcontaining ampicillin. After overnight culture at 37° C., the culturesare then spun down by centrifugation. Individual cell aliquots are thenretested for binding to the target ligand attached to the beads. Bindingto other beads, having attached thereto a non-relevant ligand, can beused as a negative control.

One important aspect of screening the libraries is that of elution. Forclarity of explanation, the following is discussed in terms of TSARexpression by phage; however, it is readily understood that suchdiscussion is applicable to any system where the random peptide isexpressed on a surface fusion molecule. It is conceivable that theconditions that disrupt the peptide-target interactions during recoveryof the phage are specific for every given peptide sequence from aplurality of proteins expressed on phage. For example, certaininteractions may be disrupted by acid pH's but not by basic pH's, andvice versa. Thus, it may be desirable to test a variety of elutionconditions (including but not limited to pH 2-3, pH 12-13, excess targetin competition, detergents, mild protein denaturants, urea, varyingtemperature, light, presence or absence of metal ions, chelators, etc.)and compare the primary structures of the TSAR proteins expressed on thephage recovered for each set of conditions to determine the appropriateelution conditions for each ligand/TSAR combination. Some of theseelution conditions may be incompatible with phage infection because theyare bactericidal and will need to be removed by dialysis (i.e., dialysisbag, Centricon/Amicon microconcentrators).

The ability of different expressed proteins to be eluted under differentconditions may not only be due to the denaturation of the specificpeptide region involved in binding to the target but also may be due toconformational changes in the flanking regions. These flanking sequencesmay also be denatured in combination with the actual binding sequence;these flanking regions may also change their secondary or tertiarystructure in response to exposure to the elution conditions (i.e., pH2-3, pH 12-13, excess target in competition, detergents, mild proteindenaturants, urea, heat, cold, light, metal ions, chelators, etc.) whichin turn leads to the conformational deformation of the peptideresponsible for binding to the target.

According to another alternative method in which the TSARs contain alinker region between the binding domain and the effector domain,particular TSAR libraries can be prepared and screened by: (1)engineering a vector, preferably a phage vector, so that a DNA sequenceencodes a segment of Factor Xa (or Factor Xa protease cleavable peptide)and is present adjacent to the gene encoding the effector domain, e.g.,the pIII coat protein gene; (2) construct and assemble the doublestranded synthetic oligonucleotides as described above and insert intothe engineered vector; (3) express the plurality of vectors in asuitable host to form a library of vectors; (4) screen for binding to animmobilized ligand; (5) wash away excess phage; and (6) treat theimmobilized phage with Factor Xa protease. The particle will beuncoupled from the peptide-ligand complex and can then be used to infectbacteria to regenerate the particle with its full-length pIII moleculefor additional rounds of screening. This alternative embodimentadvantageously allows the use of universally effective elutionconditions and thus allows identification of phage expressing TSARs thatotherwise might not be recovered using other known methods for elution.To illustrate, using this embodiment, exceptionally tight binding TSARscould be recovered. If desired, the oligonucleotides encoding thebinding domain selected in this manner can be mutagenized, expressed andrechromatographed (or screened by another method) to discover improvedbinding activity. In particular, saturation mutagenesis can be carriedout using synthetic oligonucleotides synthesized from "doped" nucleotidereservoirs. The doping is carried out such that the original peptidesequence is represented only once in 10⁶ unique clones of themutagenized oligonucleotide. The assembled oligonucleotides are clonedinto a parental TSAR vector. Preferably, the vector is m663 (Fowlkes etal., 1992, BioTechniques 13:422-427). m663 is able to make blue plaqueswhen grown in E. coli stain JM101 or DH5αF'. A library of greater than10⁶ is preferred; however a library of 10⁵ is sufficient to isolate TSARphage displaying peptide domains with increased selectivity for bindingto the target ligand.

For the examples in Section 6 herein, a TSAR library is utilized;however, to those skilled in the art, it will be apparent that otherpeptide libraries may be used. An example of a TSAR library is theTSAR-9 library disclosed in Kay et al., 1993, Gene 128:59-65. TSAR-9constructs display a peptide of about 38 amino acids in length having 36totally random positions.

5.3.1. Antibodies and Derivatives Thereof For Use in Screening

Antibodies can be produced which recognize an antigen of interest. Suchantibodies can be polyclonal or monoclonal. Such antibodies may be usedas ligands in the first screening step of the present invention.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to an antigen of interest. For the production ofantibody, various host animals can be immunized by injection with anantigen of interest or derivative thereof, including but not limited torabbits, mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, and including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum. A

monoclonal antibody to an antigen of interest can be prepared by usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Kohler and Milstein(1975, Nature 256:495-497), and the more recent human B cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72) and EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96).

The monoclonal antibodies may be human monoclonal antibodies or chimerichuman-mouse (or other species) monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312;Kozbor et al., 1983, Immunology Today 4:72-79; Olsson et al., 1982,Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be preparedcontaining a mouse antigen-binding domain with human constant regions(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851, Takeda etal., 1985, Nature 314:452).

A molecular clone of an antibody to an antigen of interest can beprepared by known techniques. Recombinant DNA methodology (see e.g.,Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.) may be used toconstruct nucleic acid sequences which encode a monoclonal antibodymolecule, or antigen binding region thereof.

Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

Antibody fragments which contain the idiotype or antigen binding regionof the molecule can be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab')2 fragment which canbe produced by pepsin digestion of the antibody molecule; the Fab'fragments which can be generated by reducing the disulfide bridges ofthe F(ab')2 fragment, and the 2 Fab or Fab fragments which can begenerated by treating the antibody molecule with papain and a reducingagent.

5.4. Identification of Synthetic Sequences Which Mediate Binding

When a peptide from a peptide library has been identified as an abtideor mimetope for a particular target ligand of interest according to themethod of the invention (in either the first or second screening step),it may be useful to determine what region(s) of the expressed peptidesequence is (are) responsible for binding to the target ligand. Suchanalysis can be conducted at two different levels, i.e., the nucleotidesequence and amino acid sequence levels.

By molecular biological techniques it is possible to verify and furtheranalyze a ligand binding peptide at the level of the oligonucleotides.First, the inserted oligonucleotides can be cleaved using appropriaterestriction enzymes and religated into the original expression vectorand the expression product of such vector screened for ligand binding toidentify the oligonucleotides that encode the binding region of theabtide or mimetope. Second, the oligonucleotides can be transferred intoanother vector, e.g., from phage to phagemid. The newly expressed fusionproteins should acquire the same binding activity if the domain isnecessary and sufficient for binding to the ligand. This last approachalso assesses whether or not flanking amino acid residues encoded by theoriginal vector influence peptide binding in any fashion. Third, theoligonucleotides can be synthesized, based on the nucleotide sequencedetermined for the phage in the library that encodes the bindingpeptide, amplified by cloning or PCR amplification using internal andflanking primers, cleaved into two pieces and cloned as two half-bindingdomain fragments. In the foregoing manner, the inserted oligonucleotidesare subdivided into two equal halves. If the peptide domain importantfor binding is small, then one recombinant clone would demonstratebinding and the other would not. If neither have binding, then eitherboth are important or the essential portion of the domain spans themiddle (which can be tested by expressing just the central region).

Alternatively, by synthesizing peptides corresponding to the deducedsequence of the abtide or mimetope, the binding domains can be analyzed.First, the entire peptide should be synthesized and assessed for bindingto the target ligand to verify that the peptide is necessary andsufficient for binding. Second, short peptide fragments, for example,overlapping 10-mers, can be synthesized, based on the amino acidsequence of the random peptide binding domain, and tested to identifythose binding the ligand.

In addition, in certain instances, linear motifs (consensus motifs) maybecome apparent after comparing the primary structures of differentbinding peptides from the library having binding affinity for a targetligand. The contribution of these motifs to binding can be verified withsynthesized peptides in competition experiments (i.e., determine theconcentration of peptide capable of inhibiting 50% of the binding of thephage to its target; IC₅₀). Conversely, the motif or any regionsuspected to be important for binding can be removed from or mutatedwithin the DNA encoding the random peptide insert and the altereddisplayed peptide can be retested for binding.

Furthermore, once the binding domain of a peptide has been identified,the binding characteristics of that peptide can be modified by varyingthe binding domain sequence to produce a related family of peptides withdiffering properties for a specific ligand.

Moreover, in a method of directed evolution, the identified peptides canbe improved by additional rounds of random mutagenesis, selection, andamplification of the nucleotide sequences encoding the binding domains.Mutagenesis can be accomplished by creating and cloning a new set ofoligonucleotides that differ slightly from the parent sequence, e.g., by1-10%. Selection and amplification are achieved as described above. Byway of example, to verify that the isolated peptides have improvedbinding characteristics, mutants and the parent phage, differing intheir lacZ expression, can be processed together during the screeningexperiments. Alteration of the original blue-white color ratios duringthe course of the screening experiment will serve as a visual means toassess the successful selection of enhanced binders. This process can gothrough numerous cycles.

5.5. Uses of Abtides 5.5.1. Assays Using Abtides

The abtides of the present invention possess binding specificities thatare similar to those of the ligands (e.g. antibodies, receptors) thatare used in the first screening step of the process by which the abtidesare identified. Consequently, the abtides may be used in many of thesame instances where the ligand of the first screening step might beused. For example, if the ligand used in the first screening step is anantibody, the abtide that is identified after the second screening stepwill bind specifically to the same antigen to which the antibodyspecifically binds. Therefore, the abtide may be used as a substitutefor the antibody in many of the reactions or assays that the antibodycould be used in. For example, the abtide could be used in immunoassaysknown in the art, e.g., those designed to detect or measure the amountof the antigen. Of course, such immunoassays may have to be suitablymodified. For example, many immunoassays make use of a step in which asecond antibody, labeled with a radioactive moiety or an enzyme such asalkaline phosphatase, specifically binds to the first antibody. Such asecond antibody would not be expected to specifically bind to theabtide. However, it would be well within the competence of one ofordinary skill in the art to fabricate another labelling moiety, perhapsa third antibody, that was able to specifically bind to the abtide, orto label the abtide with a detectable marker prior to use.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassays), "sandwich" immunoassays, dot immunoblot assays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, immunoaffinity chromatography, andflow dipstick assays to name but a few. For examples of exemplaryprocedures which can be used in immunoassays, see generally Kricka,1985, Clinical and Biochemical Analysis 17:1-15; Armbruster, 1993, Clin.Chem. 39/2:181-195; Birnbaum et al., 1992, Anal. Biochem. 206:168-171;Miyai, 1985, Adv. Clin. Chem. 24:61-110; and references cited therein.

The samples to be assayed in the immunoassays can be any sample that maycontain the antigen or ligand desired to be assayed. For example, thesesamples can be body fluids such as plasma, blood, serum, saliva,cerebrospinal fluid, synovial fluid, etc.

The detectable label to be used in the immunoassays can be anydetectable label known in the art. Such labels include radioisotopes,fluorescent dyes, enzymes (for example horseradish peroxidase oralkaline phosphatase), chemiluminescent molecules, metal atoms, orphosphorescent dyes, colored particles, metal and dye colloids.

5.5.1.1. Sandwich Elisa

In a particular embodiment, the abtides can be used in a sandwich enzymeimmunoassay. One description of such an embodiment, presented by way ofexample and not limitation, follows: A molecule comprising an abtide isaffixed to a solid substratum. The molecule comprising the abtide may belinked to a substance that will provide for greater attachment of themolecule to the substratum. The sample to be assayed is contacted withthe molecule comprising the abtide that is bound to the substratum. Thesubstances in the sample that are specific binding partners of theabtide (analyte) will bind to the abtide, and non-binding samplecomponents are removed by washing. An enzyme-conjugated monoclonalantibody directed against the analyte is added. This enzyme-conjugatedmonoclonal antibody binds to the part of the analyte it is specific forand completes the sandwich. After removal of unbound enzyme-conjugatedmonoclonal antibody by washing, a substrate solution is added to thewells. A colored product is formed in proportion to the amount ofanalyte present in the sample. The reaction may be terminated byaddition of stop solution and absorbance is measuredspectrophotometrically. The following illustrates these steps in moredetail.

(a) Polystyrene microtiter wells (Flow Laboratory) are coated overnightat room temperature with 100 μl of a solution of a molecule comprisingan abtide at a concentration of 1 mg/ml in phosphate buffered saline(PBS).

(b) Coating solution is discarded and wells are blocked for 1-2 hours atroom temperature with 300 μl of 1% bovine serum albumin (BSA) inphosphate-buffered saline (PBS) with 0.05% of TWEEN™ 20(polyoxyethylenesorbitan monolaurate) 20 (PBS-TWEEN™ buffer).

(c) 150 μl of sample (suspected of containing an analyte the presence oramount of which it is desired to measure) diluted in 1% BSA-PBS is addedper well. Wells are incubated 1 hour at room temperature.

(d) Wells are washed 4 times with PBS-TWEEN™ buffer.

(e) 100 μl of horseradish peroxidase conjugated monoclonal antibodyspecific for the analyte in 1% BSA-PBS is added per well. Theconcentration of the monoclonal antibody can be from about 10 ng/ml to10 mg/ml. Wells are incubated 1 hour at room temperature.

(f) Wells are washed 6 times with PBS-TWEEN™ buffer.

(g) 100 μl of ABTS® Boehringer Mannheim(2,2'-Azino-di-[3-ethylbenzthiazdine sulfonate (6)] crystallizeddiammonium salt working solution is added per well. ABTS® stock solutionis prepared at 15 mg/ml in dH₂ O. To make the working solution, 200 μlof this ABTS® stock is diluted into 10 ml of citrate phosphate buffer(17 mm citric acid, 65 mm dibasic sodium phosphate) and 10 μl 30% H₂ O₂.

(h) The absorbance of each well is measured at 405 nm in a microtiterplate reader (Dynatech MR600, Dynatech Corp., Alexandria, Va.).

5.5.2. Pharmaceutical Compositions

The invention provides methods of treatment by administration to asubject of an effective amount of a pharmaceutical (therapeutic ordiagnostic) composition comprising an abtide. Such an abtide envisionedfor therapeutic or diagnostic use is referred to hereinafter as a"Therapeutic" or "Therapeutic of the invention." Such therapeutics areabtides that specifically bind to a molecule in vivo, to exert atherapeutic or diagnostic effect. In a preferred aspect, the Therapeuticis substantially purified. The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed areknown in the art and can be selected from among those describedhereinbelow.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells containing theTherapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes as wellas transdermal and subcutaneous time-release implants. The Therapeuticsmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. In a specificembodiment, it may be desirable to utilize liposomes targeted viaabtides to specific identifiable cell surface antigens.

In a specific embodiment, it may be desirable to administer theTherapeutics of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

The present invention provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.5.3. In Vivo Diagnostic and Therapeutic Uses of Abtides

Another area where abtides can be used in place of antibodies is in theimaging, detection, or treatment of disease. Current diagnostic andtherapeutic methods make use of antibodies to target imaging agents ortherapeutic substances, e.g. to tumors. Since abtides possess the samespecificity of binding as antibodies, abtides can be used in place ofantibodies in such diagnostic and therapeutic methods.

Abtides may be linked to chelators such as those described in U.S. Pat.No. 4,741,900 or U.S. Pat. No. 5,326,856. The abtide-chelator complexmay then be radiolabeled to provide an imaging agent for diagnosis ortreatment of disease. The abtide may also be used in the methods thatare disclosed in co-pending U.S. patent application Ser. No. 08/127,351now U.S. Pat. No. 3,449,761 for creating a radiolabeled peptide for usein imaging or radiotherapy. This application contains a review ofmethods of using peptides in imaging agents.

In in vivo diagnostic applications, specific tissues or even specificcellular disorders may be imaged by administration of a sufficientamount of a labeled abtide of the instant invention.

A wide variety of metal ions suitable for in vivo tissue imaging havebeen tested and utilized clinically. For imaging with radioisotopes, thefollowing characteristics are generally desirable: (a) low radiationdose to the patient; (b) high photon yield which permits a nuclearmedicine procedure to be performed in a short time period; (c) abilityto be produced in sufficient quantities; (d) acceptable cost; (e) simplepreparation for administration; and (f) no requirement that the patientbe sequestered subsequently. These characteristics generally translateinto the following: (a) the radiation exposure to the most criticalorgan is less than 5 rad; (b) a single image can be obtained withinseveral hours after infusion; (c) the radioisotope does not decay byemission of a particle; (d) the isotope can be readily detected; and (e)the half-life is less than four days (Lamb and Kramer, "CommercialProduction of Radioisotopes for Nuclear Medicine", In Radiotracers ForMedical Applications, Vol. 1, Rayudu (Ed.), CRC Press, Inc., Boca Raton,pp. 17-62). Preferably, the metal is technetium-99m.

By way of illustration, the targets that one may image include any solidneoplasm, certain organs such a lymph nodes, parathyroids, spleen andkidney, sites of inflammation or infection (e.g., macrophages at suchsites), myocardial infarction or thromboses (neoantigenic determinantson fibrin or platelets), and the like evident to one of ordinary skillin the art. Furthermore, the neoplastic tissue may be present in bone,internal organs, connective tissue, or skin.

As is also apparent to one of ordinary skill in the art, one may use thepresent invention in in vivo therapeutics (e.g., using radiotherapeuticmetal complexes), especially after having diagnosed a diseased conditionvia the in vivo diagnostic method described above, or in in vitrodiagnostic application (e.g., using a radiometal or a fluorescent metalcomplex).

Accordingly, a method of obtaining an image of an internal region of asubject is contemplated in the instant invention which comprisesadministering to a subject an effective amount of an abtide compositioncontaining a metal in which the metal is radioactive, and recording thescintigraphic image obtained from the decay of the radioactive metal.Likewise, a method is contemplated of enhancing an MR image of aninternal region of a subject which comprises administering to a subjectan effective amount of an abtide composition containing a metal in whichthe metal is paramagnetic, and recording the MR image of an internalregion of the subject.

Other methods include a method of enhancing a sonographic image of aninternal region of a subject comprising administering to a subject aneffective amount of an abtide composition containing a metal andrecording the sonographic image of an internal region of the subject. Inthis latter application, the metal is preferably any non-toxic heavymetal ion. A method of enhancing an X-ray image of an internal region ofa subject is also provided which comprises administering to a subject anabtide composition containing a metal, and recording the X-ray image ofan internal region of the subject. A radioactive, non-toxic heavy metalion is preferred.

The use of abtides in place of antibodies in such methods has certainadvantages. Because abtides are peptides rather than large proteins suchas antibodies, the kinetics of their distribution in the body andclearance from the bloodstream differ from that of large proteins suchas antibodies. For example, as demonstrated in Section 6.1.4, abtidescan be used for in vivo imaging of disease states in about 2 to 5 hours.Current methods of tumor imaging using antibodies require approximately24 to 48 hours.

Because abtides are peptides, they are cleared from the blood fasterthan antibodies. This means that there will be less background signal inthe bloodstream when using abtides to image disease states than there iswhen using antibodies.

Peptides most likely will provoke less of an immune response in patientsthan do large proteins such as antibodies. This consideration isespecially important when diagnosis or treatment is required to be donerepeatedly or over a long period of time.

Abtides, because they are generally small proteins, can remain solublein physiological fluids under conditions where antibodies cannot.

Abtides, again because they are generally peptides, can be producedsynthetically or by recombinant methods and therefore may be less costlyto produce than antibodies.

Abtides may be used individually. Alternatively, abtides may be used ascompositions of abtides in which the peptide sequences of the abtidesdiffer.

5.6. Synthesis of Peptides 5.6.1. Procedure for Solid Phase Synthesis

Abtide or mimetope peptides may be prepared by methods that are known inthe art. For example, in brief, solid phase peptide synthesis consistsof coupling the carboxyl group of the C-terminal amino acid to a resinand successively adding N-alpha protected amino acids. The protectinggroups may be any known in the art. Before each new amino acid is addedto the growing chain, the protecting group of the previous amino acidadded to the chain is removed. The coupling of amino acids toappropriate resins is described by Rivier et al., U.S. Pat. No.4,244,946. Such solid phase syntheses have been described, for example,by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981,Science 213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178and in U.S. Pat. Nos. 4,305,872 and 4,316,891. In a preferred aspect, anautomated peptide synthesizer is employed.

By way of example but not limitation, peptides can be synthesized on anApplied Biosystems Inc. ("ABI") model 431A automated peptide synthesizerusing the "Fastmoc" synthesis protocol supplied by ABI, which uses2-(1H-Benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate("HBTU") (R. Knorr et al., 1989, Tet. Lett., 30:1927) as coupling agent.Syntheses can be carried out on 0.25 mmol of commercially available4-(2',4'-dimethoxyphenyl-(9-fluorenyl-methoxycarbonyl)-aminomethyl)-phenoxypolystyrene resin ("Rink resin" from Advanced ChemTech) (H. Rink, 1987,Tet. Lett. 28:3787). Fmoc amino acids (1 mmol) are coupled according tothe FASTMOC protocol. The following side chain protected Fmoc amino acidderivatives are used: FmocArg(Pmc)OH; FmocAsn (Mbh) OH; FmocAsp (^(t)Bu) OH; FmocCys (Acm) OH; FmocGlu(^(t) Bu)OH; FmocGln(Mbh)OH;FmocHis(Tr)OH; FmocLys(Boc)OH; FmocSer (^(t) Bu) OH; FmocThr (^(t) Bu)OH; FmocTyr (^(t) Bu) OH. [Abbreviations: Acm, acetamidomethyl; Boc,tert-butoxycarbonyl; ^(t) Bu, tert-butyl; Fmoc,9-fluorenylmethoxycarbonyl; Mbh, 4,4'-dimethoxybenzhydryl; Pmc,2,2,5,7,8-pentamethylchroman-6-sulfonyl; Tr, trityl].

Synthesis is carried out using N-methylpyrrolidone (NMP) as solvent,with HBTU dissolved in N,N-dimethylformamide (DMF). Deprotection of theFmoc group is effected using ca. 20% piperidine in NMP. At the end ofeach synthesis the amount of peptide present is assayed by ultravioletspectroscopy. A sample of dry peptide resin (ca. 3-10 mg) is weighed,then 20% piperidine in DMA (10 mL) is added. After 30 min sonication,the UV (ultraviolet) absorbance of the dibenzofulvene-piperidine adduct(formed by cleavage of the N-terminal Fmoc group) is recorded at 301 nm.Peptide substitution (in mmol g⁻¹) can be calculated according to theequation: ##EQU1## where A is the absorbance at 301 nm, v is the volumeof 20% piperidine in DMA (in mL), 7800 is the extinction coefficient (inmol⁻¹ dm³ cm⁻¹) of the dibenzofulvene-piperidine adduct, and w is theweight of the peptide-resin sample (in mg).

Finally, the N-terminal Fmoc group is cleaved using 20% piperidine inDMA, then acetylated using acetic anhydride and pyridine in DMA. Thepeptide resin is thoroughly washed with DMA, CH₂ Cl₂ and finally diethylether.

5.6.2. Cleavage and Deprotection

By way of example but not limitation, cleavage and deprotection can becarried out as follows: The air-dried peptide resin is treated withethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole(PhSMe) for approximately 20 min. prior to addition of 95% aqueoustrifluoracetic acid (TFA). A total volume of ca. 50 mL of these reagentsare used per gram of peptide-resin. The following ratio is used:TFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5). The mixture is stirred for 3 h atroom temperature under an atmosphere of N₂. The mixture is filtered andthe resin washed with TFA (2×3 mL). The combined filtrate is evaporatedin vacuo, and anhydrous diethyl ether added to the yellow/orangeresidue. The resulting white precipitate is isolated by filtration. SeeKing et al., 1990, Int. J. Peptide Protein Res. 36:255-266 regardingvarious cleavage methods.

5.6.3. Purification of the Peptides

Purification of the synthesized peptides can be carried out by standardmethods including chromatography (e.g., ion exchange, affinity, andsizing column chromatography, high performance liquid chromatography(HPLC)), centrifugation, differential solubility, or by any otherstandard technique.

5.6.4. Conjugation of Peptides to Other Molecules

The abtides of the present invention may be linked to other molecules(e.g., a detectable label, a molecule facilitating adsorption to a solidsubstratum, or a toxin, according to various embodiments of theinvention) by methods that are well known in the art. Such methodsinclude the use of homobifunctional and heterobifunctional cross-linkingmolecules.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., 1984, Science 223:1304-1306.

Homobifunctional molecules having at least two active ester unitsinclude esters of dicarboxylic acids and N-hydroxysuccinimide. Someexamples of such N-succinimidyl esters include disuccinimidyl suberateand dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonicacid and bis-sulfonate salts such as their sodium and potassium salts.These homobifunctional reagents are available from Pierce, Rockford,Ill.

The heterobifunctional molecules have at least two different reactivegroups. Some examples of heterobifunctional reagents containing reactivedisulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate(Carlsson et al., 1978, Biochem J. 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-5N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce.

Additional information regarding how to make and use these as well asother polyfunctional reagents may be obtained from the followingpublications or others available in the art:

Carlsson et al., 1978, Biochem. J. 173:723-737.

Cumber et al., 1985, Methods in Enzymology 112:207-224.

Jue et al., 1978, Biochem 17:5399-5405.

Sun et al., 1974, Biochem. 13:2334-2340.

Blattler et al., 1985, Biochem. 24:1517-152.

Liu et al., 1979, Biochem. 18:690-697.

Youle and Neville, 1980, Proc. Natl. Acad. Sci. USA 77:5483-5486.

Lerner et al., 1981, Proc. Natl. Acad. Sci. USA 78:3403-3407.

Jung and Moroi, 1983, Biochem. Biophys. Acta 761:162.

Caulfield et al., 1984, Biochem. 81:7772-7776.

Staros, 1982, Biochem. 21:3950-3955.

Yoshitake et al., 1979, Eur. J. Biochem. 101:395-399.

Yoshitake et al., 1982, J. Biochem. 92:1413-1424.

Pilch and Czech, 1979, J. Biol. Chem. 254:3375-3381.

Novick et al., 1987, J. Biol. Chem. 262:8483-8487.

Lomant and Fairbanks, 1976, J. Mol. Biol. 104:243-261.

Hamada and Tsuruo, 1987, Anal. Biochem. 160:483-488.

Hashida et al., 1984, J. Applied Biochem. 6:56-63.

Additionally, methods of cross-linking are reviewed by Means and Feeney,1990, Bioconjugate Chem. 1:2-12.

5.6.4.1. Biotinylation of Peptides

Methods of biotinylating peptides are well known in the art. Anyconvenient method may be employed in the practice of the invention. Forexample, the following procedure was used:

(1) dissolve 10 mg of peptide in 100 μL of 0.1% acetic acid;

(2) add 900 μL of PBS;

(3) add 3.3 mg of biotin-LC-NHS (Pierce, Rockford, Ill.);

(4) incubate for 30 minutes at room temperature;

(5) purify over a Superose 12 column (Pharmacia, Piscataway, N.J.).

6. EXAMPLES 6.1. Abtides Mimicking the Binding Specificity of MonoclonalAntibody 7E11-C5 6.1.1. Identification and Isolation of AbtidesMimicking the Binding Specificity of Monoclonal Antibody 7E11-C5

7E11-C5 is a murine IgG1 monoclonal antibody specific for an antigen ofa human prostate carcinoma, LNCaP. 7E11-C5 binds strongly to malignantprostatic epithelium but only weakly to normal prostatic epithelium. Itdoes not bind to non-prostatic tumors or to most normal organs. SeeHoroszewicz et al., 1987, Anticancer Res. 7:927-936. 7E11-C5 is alsodescribed in U.S. Pat. No. 5,162,504 issued Nov. 10, 1992. Hybridomasproducing monoclonal antibody 7E11-C5 were grown as ascites in mice and7E11-C5 was purified from ascites fluid by Protein A affinitychromatography to over 90% purity as judged by sodium dodecylsulfatepolyacrylamide gel electrophoresis.

In order to identify abtides mimicking binding specificity of monoclonalantibody 7E11-C5, monoclonal antibody 7E11-C5 was used as the targetligand in a first screening of the TSAR-9 library (see Kay et al., 1993,Gene 128:59-65 and PCT publication WO 94/18318, dated Aug. 18, 1994).The following screening procedure was used. First, 7E11-C5 was bound toa well of a microtiter plate. 7E11-C5 at a concentration of 11.2 mg/mLin phosphate buffered saline (PBS), pH 6.0, was diluted to 100 μg per mLin 0.1× PBS pH 7.2. One hundred microliters (100 μL) of this dilutionwas added to one well of a microtiter plate, and allowed to incubate for1-6 hours at room temperature or overnight at 4° C. After incubation,the well was washed at least 4 times with a blocking buffer whichconsisted of either 1% bovine serum albumin (BSA) in PBS, 1% non-fat drymilk (NFDM) in PBS, or 0.1% TWEEN® in either it BSA in PBS or 1% NFDM inPBS. Two hundred microliters of the blocking buffer was then added tothe well and allowed to incubate for at least an hour at roomtemperature.

Next, an aliquot of the TSAR-9 library was added to the well containingbound 7E11-C5. An aliquot of the library containing 10¹⁰ phage particleswas added to the well and allowed to incubate for at least 1 hour atroom temperature. This resulted in the binding to the plate of thosephage containing binding domains that bind to 7E11-C5. After an hour,the well was washed extensively with either 1% bovine serum albumin(BSA) in PBS, 1% non-fat dry milk (NFDM) in PBS, or 0.1% TWEEN® ineither 1% BSA in PBS or 1% NFDM in PBS.

After washing, phage bound to the 7E11-C5 antibody in the well wereeluted by adding 100 μL of an acid solution of 0.2 M glycine-HCl, pH2.0. After incubation from 15 minutes to 1 hour, the acid solutioncontaining eluted phage was transferred to a 1.5 mL microfuge tube, andan equal volume of 0.2 M Tris-HCl, pH 7.5 added to neutralize the acidsolution. In some cases, the neutralized phage solution was immediatelyadded to a second well containing bound 7E11-C5 antibody, and thebinding and elution procedure repeated.

If it was desired that the level of enrichment be monitored during theabove steps, an irrelevant phage that does not bind 7E11-C5 but thatexpresses the β-galactosidase gene was added to the aliquot from theTSAR-9 library. This phage gives rise to blue plaques when plated in thepresence of X-Gal and IPTG. Following a screening step, the eluted phagewere plated in X-gal and IPTG. An aliquot of unscreened phage wereplated as well. The ratio of white to blue plaques was measured for bothphage samples. The increase in the proportion of white plaques (from theTSAR-9 phage that bind to 7E11-C5) to blue plaques (from the irrelevantphage) indicated the degree to which the screening process enriched thepopulation of phage for those phage that bind 7E11-C5.

If it was desired that the specificity of binding be monitored duringthe above screening steps, screening was done against an irrelevanttarget (either BSA, mouse IgG, or plastic) in addition to being doneagainst 7E11-C5. The enrichment of white plaques over blue plaques whenpanning was done against 7E11-C5 rather than an irrelevant targetindicated the level of specificity of binding.

After screening, the phage were amplified by adding an aliquot of theeluted phage to a solution containing LB broth and competent DH5αF' E.coli cells (GIBCO BRL, Gaithersburg, Md.). Typically a 2-5 μL aliquot ofthe phage solution was added to 125 μL of LB broth containing a 1:50dilution of DH5αF' E. Coli cells (about 1×10⁹ cells/ml). 3.3 mL of topagar was added to this solution, and the mixture was plated out onto aPetri dish containing agar. Often the phage were titered by makingseveral serial 1:10 dilutions, and plating out the dilution as describedabove. After incubation overnight at 37° C., the plates were evaluatedfor growth of plaques, and counted if desired. The plates were eluted byadding 3-5 mL of 100 mM NaCl, 10 mM MgCl₂, 10 mM Tris-HCl, pH 7.5 (SMbuffer) to each plate and incubating for 1-5 hours with gentle rocking.The solution was then removed from the plate, centrifuged, and eitherstored, amplified further, or analyzed.

In some cases, the phage were amplified in solution by adding 1-5 μL ofthe phage solution to 1-5 mL of LB broth containing a 1:50 or 1:100dilution of competent DH5αF' E. coli cells (about 1×10⁹ cells/ml. Afterincubation for either 6 hours or overnight, the solution was centrifugedand the supernatant collected In some cases, the phage particles wereprecipitated with polyethylene glycol (PEG) by adding a 1/5 volume ofPEG to the clarified phage solution, and incubating for 1 hour on ice.After centrifugation, the phage were usually reconstituted with 100 μLof SM buffer.

Using the above procedures, nine different phage were isolated thatexpressed peptides containing binding domains that were capable ofbinding monoclonal antibody 7E11-C5. Molecules comprising these bindingdomains are thus mimetopes of the antigen recognized by the monoclonalantibody 7E11-C5. The binding domains of the peptides expressed by thenine phage were sequenced according to standard methods of DNAsequencing (SEQUENASE™, U.S. Biochemical Corp., Cleveland, Ohio). Thedetermination of those DNA sequences allowed the determination of theamino acid sequences of these mimetopes. These sequences are shown inTable 1. Examination of these amino acid sequences showed that theyshared a common motif of MYxxLH (SEQ ID NO. 10).

                                      TABLE 1                                     __________________________________________________________________________    SCVSHMLDTSRVYTAYANPG                                                                            MYSRLH SPAVRPLTQSSA     (SEQ ID No.: 11)                       - SVQFKSISSRSMDDVVKDPGPKPA      MYNRLH SKNPFTLS                                                                      (SEQ ID No.: 12)                       - YFDHTYSGPVVKNGGLVSPGVLS      MYNRLH SDGGPSLAS                                                                      (SEQ ID No.: 13)                       - TVAT       MHDRLH SAPGSGNLPGSYDIKPIFKAQSGALHS  (SEQ ID No.: 14)                                                      -   T                                - IDMPQTAST      MYNMLH RNEPGGRKLSPPANDMPPALLKR         (SEQ ID No.:                                                 15)                                    - RLGNHVWREGGG      MYQQLH HNFP                         (SEQ ID No.:                                                 16)                                    - RDSAVENPSVGGEIP      MYRYLH QR                           (SEQ ID                                                   No.: 17)                               - PVQKEYGFGNSGAS      MIRLLR ETP                          (SEQ ID No.:                                               18)                                    - QKGGPGLLLYGGDS      MYITLH EPG                          (SEQ ID No.:                                               19)                                    -       MYxxLH                               (SEQ ID No.: 10)                 -  LYANPGMYSRLHSPA                          (SEQ ID NO: 20)                __________________________________________________________________________

In order to use the mimetopes to identify abtides, peptidescorresponding to the mimetope sequences were a final concentration of 5μg/mL. Specifically, a peptide called 7E11-9.5, with the sequenceLYANPGMYSRLHSPA (SEQ ID NO: 20) and a peptide with the sequenceGMYSRLHSPA (SEQ ID NO: 21) were synthesized.

First the mimetope peptide 7E11-9.5 was tested for its ability to bindto the monoclonal antibody 7E11-C5. Ninety-six well plates (Immunlon 4,Dynatech, Alexandria, Va.) were coated with 50 μL of a 5 μg/mL solutionof the mimetope peptide 7E11-9.5 and incubated overnight at roomtemperature. Following incubation, the wells were washed 4 times with 1%BSA in PBS. Biotinylated monoclonal antibody 7E11-C5 was seriallydiluted with PBS beginning with a concentration of 29 μg/mL and variousamounts of the monoclonal antibody were added to the wells that had beencoated with the mimetope peptide. After incubating for 1 hour at roomtemperature, the wells were washed four times with 1% BSA in PBS and 2times with PBS. Then, 100 μL of a 1:2000 dilution of Extravidin-AlkalinePhosphatase (4,250 units/mL) (Sigma, St. Louis, Mo.) in PBS was added toeach well. After an hour, the plate was again washed 4 times with PBSand 100 μL of a 1 mg/mL solution of the enzyme substrate p-nitrophenylphosphate was added to each well. Color was allowed to develop for 15minutes to 1 hour and absorbance was read at 405 nm. FIG. 2 shows theresults. It can be seen from FIG. 2 that monoclonal antibody 7E11-C5binds to the synthesized mimetope 7E11-9.5 in a concentration dependentmanner.

Next, the mimetope peptide was used to isolate abtides from a peptidelibrary. Fifty to one hundred microliters of the solution of thispeptide was used to coat the wells of a 96-well plate, the wells wereblocked with 0.5% BSA in PBS, and the wells were used for screening. Analiquot of the TSAR-9 random phage library (containing approximately3×10¹⁰ phage particles) was used as in the initial screening, and 4rounds of screening were performed. After the first two rounds, thephage were amplified. Two more rounds of screening were then performed.By this procedure, phage from the TSAR-9 library that expressed peptidescapable of binding to the 7E11-9.5 mimetope peptide were identified andisolated. The peptides containing the binding domains of these phage areabtides and were discovered to mimic the binding specificities ofmonoclonal antibody 7E11-C5. These abtides are termed "7E11-C5 abtides."

Phage encoding the 7E11-C5 abtides were subjected to DNA sequencing ofthe nucleotide sequences encoding their binding peptides in order toobtain the DNA and amino acid sequences of the 7E11-C5 abtides. Table 2shows the amino acid sequence of five of the 7E11-C5 abtides that hadrelatively high affinity for the mimetope.

                  TABLE 2                                                         ______________________________________                                        Clone   Sequence                                                              ______________________________________                                        14      GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG                                   - 17  DLSRNLDFGRFLLYNAYVPGFTPTFISLTAEHLSSPKG                                  - 15  CGRAYCLSGNYNIFGALFPGVSTPYADVGHDDAQSWRR                                  - 13  RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT                                  - 16  GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA                               ______________________________________                                    

The amino acid sequence shown in Table 2 for clone 14 is SEQ ID NO: 1.The amino acid sequence shown in Table 2 for clone 17 is SEQ ID NO: 2.The amino acid sequence shown in Table 2 for clone 15 is SEQ ID NO: 3.The amino acid sequence shown in Table 2 for clone 13 is SEQ ID NO: 4.The amino acid sequence shown in Table 2 for clone 16 is SEQ ID NO: 5.

It was of interest to determine whether there might be some structuralbasis for the similarity in binding characteristics between themonoclonal antibody 7E11-C5 and the 7E11-C5 abtides. The amino acidsequences of the complementarity determining regions (CDRs) of themonoclonal antibody 7E11-C5 were determined by sequencing cDNA clones ofthe genes encoding the variable regions of the antibody. These CDRs areresponsible for the specific binding of monoclonal antibody 7E11-C5 toits antigen. FIG. 3 presents a comparison of the amino acid sequences ofthe abtides of Table 2 and portions of the amino acid sequences of someof the CDRs of monoclonal antibody 7E11-C5. Surprisingly, it can be seenthat there are similarities in the sequences of these abtides and thesequences of the CDRs of the monoclonal antibody.

6.1.2. Characterization of 7E11-C5 Abtides

7E11-C5 abtides were tested for their ability to bind to the 7E11-C5mimetopes that were used as target ligands in the second screening stepabove. The DNA sequences of the regions of the phage DNA encoding theabtides were determined. This allowed the determination of the aminoacid sequences of the abtides. Based upon these determined amino acidsequences, synthetic peptides corresponding to these sequences weremade. These synthetic peptides (7E11-C5 abtides) were 38 amino acids inlength.

6.1.2.1. Dot Blots Using 7E11-C5 Abtides

In some cases, these abtides were used in a dot blot experiment. Inthose cases, 1 μL of a 1 mg/mL solution of the 38-residue abtides wasspotted onto nitrocellulose (0.2 μm or 0.45 μm, Schleicher & Schuell,Keene, N.H.) strips or circles. After drying (about 1/2 hour), thenitrocellulose was blocked for 1 hour in a solution of 1t BSA in PBS.The nitrocellulose was then allowed to incubate in approximately 5 mL ofa solution of 0.1 mg/mL of a biotinylated 7E11-9.5 mimetope peptide(biotin-LYANPGMYSRLHSPA) (SEQ ID NO: 20). This mimetope peptide was oneof those described in Section 6.1.1 above that were synthesized basedupon the nine peptides that were identified in the screening of Section6.1.1 above. After an hour, the nitrocellulose was washed approximately5 times with a solution of 1% BSA in PBS. A 1:2000 dilution ofExtravidin-Alkaline Phosphatase (4,250 units/mL) (Sigma, St. Louis, Mo.)in PBS was then added and allowed to incubate for 1 hour, after whichthe nitrocellulose was again washed extensively. Finally, a solution of5-bromo-4-chloro-3-indolyl phosphate (0.15 mg/mL) and nitro bluetetrazolium (0.3 mg/mL) (Sigma, St Louis, Mo.) (BCIP/NBT) was added asan enzyme substrate. Color was allowed to develop and the absorbance at405 nm was read.

An example of such a dot blot assay is shown in FIG. 4. In FIG. 4, the7E11-C5 abtides known as clone 14, clone 17, clone 15, clone 16, andclone 13 were tested for their ability to bind the biotinylated 7E11-9.5mimetope peptide. Also tested, as a positive control, was the monoclonalantibody 7E11-C5. 7E11-C5 was spotted onto the region marked 351 in FIG.4. Inspection of FIG. 4 shows that at least three of the abtides (clone14, clone 17, and clone 15) bound the mimetope. This shows that theseabtides are capable of mimicking the specific binding exhibited by themonoclonal antibody 7E11-C5.

6.1.2.2. 7E11-C5 Abtides in Place of Antibodies in Immunoassays

The ability of abtides synthesized having the amino acid sequenceencoded by the random inserts of the phage that bound the 7E11-9.5mimetope was further evaluated by ELISA assay methods.

The 7E11-C5 abtides clone 14 and clone 17 (See Table 2) were eachdissolved in 0.1× PBS to give a solution of 5 μg/mL. Fifty microlitersof each of these solutions was used to coat the wells of a 96-wellmicrotiter plate (Immulon 4, Dynatech, Alexandria, Va.) by overnightincubation at 4° C. Following this incubation, the abtide solutions wereremoved and the wells were blocked with 200 μL μl of a solution of 1%BSA in PBS. Mimetope peptides 7E11-9.5 (LYANPGMYSRLHSPA [SEQ IN NO: 20])and GMYSRLHSPA (SEQ ID NO: 21) were biotinylated as described in Section5.6.4.1 and dissolved in H₂ O to give 1 mg/mL solutions. Serial 1:2dilutions were made of these solutions and these dilutions were added tothe wells of the microtiter plate containing the bound abtides. Afterincubation for 1 hour at room temperature, the wells were washed fourtimes with 1% BSA in PBS. Then a 1:2000 dilution of Extravidin-AlkalinePhosphatase (4,250 units/mL) (Sigma, St. Louis, Mo.) in PBS was added toeach well and incubated for 1 hour at room temperature. Followingincubation, the wells were washed four times with 1% BSA in PBS and thentwice in PBS. One hundred microliters of a 1 mg/mL solution ofp-nitrophenyl phosphate in diethanol amine (DEA) buffer (both fromKirkegaard & Perry Laboratories, Gaithersburg, Md.) was then added and,after incubation for 15-30 minutes at room temperature, the absorbanceof the solutions in the wells was read at 405 nm. The results are shownin FIG. 5.

FIG. 5 shows that, except for the non-linear effect at highconcentrations of mimetope, there is a good correlation between theamount of mimetope added to the wells and the absorbance at 405 nm. Theuse of antibodies in assays such as enzyme-linked immunosorbent assays(ELISAs) to measure the concentration of a substance is well known inthe art. The ability of antibodies to specifically bind their antigensis crucial to the success of such assays. Since abtides alsospecifically bind to antigens, it was of interest to determine ifabtides can be used in place of antibodies in immunoassays such asELISA-like assays to measure the concentration of a substance. Theresults of FIG. 5 show that the abtides of the present invention can beused in assays in much the same way that antibodies can be used inimmunoassays such as ELISAs.

6.1.2.3. Biotinylation of Antibodies

Methods of biotinylating antibodies are well known in the art. Anyconvenient method may be employed in the practice of the invention. Forexample, the following procedure was used:

(1) dissolve 10 mg of antibody in 1 mL of PBS;

(2) add 0.44 mg of biotin-LC-NHS (Pierce, Rockford, Ill.);

(3) incubate for 30 minutes at room temperature;

(4) purify over a Superose 12 column (Pharmacia, Piscataway, N.J.).

6.1.3. 7E11-C5 Abtides and Monoclonal Antibody 7E11-C5 Recognize anLNCaP Antigen

The monoclonal antibody 7E11-C5 recognizes a prostate specific mucinantigen of the human prostate cancer cell line LNCaP (Horoszewicz etal., 1987, Anticancer Res. 7:927-936). To determine if 7E11-C5 abtidesrecognize and bind the native antigen, the following experiment wasdone.

A sandwich assay using the LNCaP antigen was performed. The wells of amicrotiter plate were coated with either monoclonal antibody 7E11-C5,the clone 14 7E11-C5 abtide, or, as a negative control, BSA. Coating andwashing was as for the assay described in Section 6.1.2.2. One hundredmicroliters of a lysate of LNCaP cells was added to the wells. The LNCaPlysate was prepared as described in PCT publication WO 94/18318, datedAug. 18, 1994. Following capture of the lysate on the plate, 100 μL of a5 μg/mL solution of biotinylated 7E11-C5 monoclonal antibody was addedto each well. Following incubation and washing as in Section 6.1.2.2, a1:2000 dilution of Extravidin-Alkaline Phosphatase (4,250 units/mL)(Sigma, St. Louis, Mo.) in PBS was added to each well and incubated for1 hour at room temperature. Following incubation, the wells were washedfour times with 1% BSA in PBS and then twice in PBS. One hundredmicroliters of a 1 mg/mL solution of p-nitrophenyl phosphate indiethanol amine (DEA) buffer (both from Kirkegaard & Perry Laboratories,Gaithersburg, Md.) was then added and, after incubation at roomtemperature for 15-30 minutes, the absorbance of the solutions in thewells was read at 405 nm.

The results are shown in FIG. 6. FIG. 6 shows that the 7E11-C5 abtide iscapable of recognizing the native 7E11-C5 antigen in LNCaP lysates. Thiswas a surprising discovery and would not have been predicted by thoseskilled in the art. It was generally felt that screening a library witha mimetope could yield a binder to that mimetope. Whether such a bindercould also bind the native epitope that the mimetope mimics was unknown.The mimetope could have represented the epitope in a loose fashion, e.g.its primary sequence could be slightly modified; its secondary structureor factors influencing the presentation of the mimetope could bedifferent from those presenting the native epitope. In such a case, thebinder to the mimetope would not be specific for the native epitope. Theforegoing is presented as possible explanation and not as a limitationof the present invention.

6.1.4. Use of 7E11-C5 Abtides in Biodistribution Studies

The 7E11 abtides described in Section 6.1 and its subsections above wereused in biodistribution studies to assess their ability to target humanprostate carcinoma LNCaP xenograft tumors that had been transplantedinto mice.

Male SCID mice (C.B-17/Icr Tac-SCID mice) were purchased from Fox Chase(Philadelphia, Pa.) or Taconic Farms (Germantown, N.Y.), were housed insterilized cages with filter bonnets, and were given autoclavedlaboratory rodent chow (Purina, St. Louis, Mo.), and filtered tap waterad libitum.

1×10⁷ cells of the human prostate tumor line LNCAP (Horoszewicz et al.,1987, Anticancer Res. 7:927-936) were injected subcutaneously (s.c.)into the left rear flank of the mice. The cells were growing inexponential phase before harvesting and had been resuspended in 0.2 mLof sterile saline. Tumors were grown in the mice for 2-3 months beforeabtides were injected into the mice.

For biodistribution studies, abtides were modified at their aminotermini with the chelator diethylene-triamine-pentaacetic acid anhydride(DTPA-A) (Sigma, St. Louis, Mo.). Approximately 2 mg of each abtide wasinitially dissolved in an appropriate volume of 0.1% acetic acid andthen 1 mL of 0.1 M sodium bicarbonate, pH 8.0, was added. Two mg ofDTPA-A was suspended in 100 μL of dimethylsulfoxide (DMSO), and 10 μL ofthe abtide solution added to this DTPA-A suspension. After 5 minincubation at room temperature, the suspension was filtered through a0.2 μm (ACRODISC polyvinylidene difluoride (PVDF) sample filter(ACRODISC, Gelman Sciences, Inc., Ann Arbor, Mich.), and purified usinga Superose-12 FPLC column (Pharmacia, Piscataway, N.J.) with PBS as therunning buffer. Modified peptides were stored-frozen at -20° C. or -70°C.

Abtides modified with DTPA were labeled with ¹¹¹ InCl₂ as follows. 0.1to 0.5 mCi of ¹¹¹ InCl₂ (Amersham, Chicago, Ill.) were first neutralizedby adding an equal volume of 0.1 M NaOAc, and then added to 100 to 200μg of the DTPA-A-modified abtide. After incubation for one half hour,the labeled peptide was purified using a Superose-12 FPLC column(Pharmacia, Piscataway, N.J.) with PBS as the running buffer. Labeledfractions were collected in a fraction collector. Tubes containing thelabeled peptide were pooled and used to prepare syringes for injectioninto mice.

In one experiment, abtide clone 14-DPTA-¹¹¹ In (see Table 2) wasinjected intravenously (i.v.) into two groups of mice bearing measurableLNCaP xenografts. About 0.2 mL of a 10 μg/mL solution of theradioactively labeled abtide in sterile saline was used. The specificactivity of the abtide was about 32 μCi/g. Thus, the total injected doseof radioactivity was about 120-140×10⁶ cpm.

The first group of mice was sacrificed 2 hours after injection of theabtide and tissues were dissected for analysis. The second group wassacrificed 4 hours after injection. Dissected tissues were weighed andthe amount of ¹¹¹ In in them was determined by gamma counting. The cpmper gram of each tissue was calculated by dividing the cpm of ¹¹¹ Infound in the tissue by the weight in grams of the tissue. The data arepresented as the ratio of the cpm/g in each organ to the cpm/g in blood(organ to blood ratio). This gave the ratios that are shown in Table 3and FIG. 7.

                  TABLE 3                                                         ______________________________________                                        BIODISTRIBUTION OF ABTIDE CLONE 14-DPTA-.sup.111 In                             IN LNCaP XENOGRAFT BEARING MICE                                                       Group 1a       Group 2b                                             Tissue.sup.c                                                                            AVG     s.e.m.     AVG    s.e.m.                                    ______________________________________                                        Blood     1.00    0.00       1.00   0.00                                        Lung                   0.81       0.04       1.06     0.26                    Spleen                 0.83       0.16       1.42     0.74                    Liver                  0.95       0.04       2.12     0.69                    Kidney-R              55.85        10.22      171.73    77.97                 Kidney-L              53.03        11.97      182.79    86.38                    Tumor                                                                                                               0.80                                                                         2.85                                   - Muscle                 0.33       0.03       0.42     0.01                 Testes-R               0.54       0.02       0.94     0.25                    Testes-L               0.68       0.24       0.88     0.24                  ______________________________________                                         .sup.a Group 1: sacrificed at 2 hours; n = 2.                                 .sup.b Group 2: sacrificed at 4 hours; n = 2.                                 .sup.c Value shown is the organ to blood ratio.                          

Table 3 and FIG. 7 show that, with the exception of kidney, the highestorgan to blood ratio is found in the tumor, both at 2 hours and at 4hours post-injection of abtide. This result shows that abtides with thebinding specificity of antibodies, e.g. that are specific for tumorantigens, can be used to localize to those tumors.

No unusual localization was seen to any non-tumor tissue or organ exceptkidney. The ratio for kidney is extremely high due to the well knowntendency of injected peptides to localize to the kidneys prior to beingcleared from the body.

In another experiment, abtide clone 17-DPTA-¹¹¹ In (see Table 2) wasinjected intravenously into four SCID mice bearing measurable LNCaPxenografts. Administration of xenografts was as above. About 0.2 mL of a0.1 μg/mL solution of the radioactively labeled clone 17 abtide insterile saline was injected. The specific activity of the abtide wasabout 2.4 μCi/ng. Thus, the total injected dose of radioactivity wasabout 100-110×10⁶ cpm.

In this experiment, mice were sacrificed at either 2 or 5 hourspost-injection with labeled abtide. Again, as above, the data arepresented as organ to blood ratios. As shown in Table 4 and FIG. 8,abtide clone 17-DPTA-¹¹¹ In localized to LNCaP xenograft tumors in mice.

                  TABLE 4                                                         ______________________________________                                        BIODISTRIBUTION OF ABTIDE CLONE                                                 17-DPTA-.sup.111 In IN LNCαP-XENOGRAFT BEARING MICE                              GROUP 1        GROUP 2                                               MOUSE #          MOUSE #                                                    TISSUE.sup.a                                                                             1       6          2     3                                         ______________________________________                                        BLOOD      1.00    1.00       1.00  1.00                                        LUNG             2.52  2.57   4.33   2.47                                     SPLEEN           5.82  3.00   5.53   3.70                                     LIVER-S          5.42  5.58   8.37   4.03                                     KIDNEY-R       235.63 234.88 321.09  106.74                                   KIDNEY-L       563.71 220.69 424.64  104.09                                   TUMOR-S         10.60 15.01  8.36   2.90                                      MUSCLE           0.93  2.96   1.16   3.04                                     TESTES-R         2.71  2.19   2.31   3.15                                     TESTES-L         1.64  1.14   5.36   3.41                                   ______________________________________                                         .sup.a Value shown is the organ to blood ratio.                          

Table 4 and FIG. 8, like Table 3 and FIG. 7, show that the injectedabtide localized to the tumor. This indicates that abtides can be usefulin the localization of tumors.

In contrast to the results of the two experiments described above, inwhich radioactively labeled abtides were shown to localize to tumors,when the same experiments were done with a radioactively labeled control(non-abtide) peptide (the tripeptide GYK-DPTA), no specific localizationto tumors was observed. This can be seen in FIG. 9, which shows thebiodistribution results for experiments using the ¹¹¹ In-labeled controlpeptide.

The radiolabeled peptide conjugate GYK-DPTA-¹¹¹ In was injectdintravenously into 5 SCID mice bearing measurable LNCaP xenografts. Micewere dissected 2 hours (n=2) and 5 hours (n=3) after injection of 1.5 μgof control peptide having a specific activity of 30 μCi/μg. The organ toblood ratios are presented in Table 5 and FIG. 9. As shown, the controlpeptide did not selectively localize to the tumor. While the tumor toblood ratio in one mouse was 3.26, the control peptide distributedequally well to other organs (e.g. lung 3.52, spleen 3.27, liver 21.70,etc.). These results show that there was non-specific uptake of thecontrol peptide in these organs. While abtide clone 14-DPTA-¹¹¹ Indemonstrated a tumor to blood ratio of only 1.85 at 2 hours (whichappears lower than that obtained with the control peptide), clone14-DPTA-¹¹¹ In demonstrated specific localization to the tumor as theorgan to blood ratios in the other organs were much lower (e.g. lung0.81, spleen 0.83, liver 0.95, etc.).

                  TABLE 5                                                         ______________________________________                                        BIODISTRIBUTION OF GYK-DPTA-.sup.111 In                                         IN LNCaP XENOGRAFT BEARING MICE                                                               GROUP 1                                                                            GROUP 2                                                       MOUSE #      MOUSE #                                                   ORGAN/BLOOD                                                                             1        2       3      4     5                                     ______________________________________                                        BLOOD      1.00     1.00    1.00   1.00  1.00                                   LUNG          2.96    3.52        0.95      2.84    2.39                      SPLEEN        1.74    3.27        0.97      2.21    4.04                      LIVER-S         31.79  21.70      21.56    25.38   17.78                      KIDNEY-R       563.87  406.27      273.82    509.53   269.30                  KIDNEY-L       584.69  417.98      297.65    433.97   280.50                     TUMOR-S                                                                                                            #STR3##                                                                       #STR4##                                                                       #STR5##                                                                       #STR6##                                                                       #STR7##                                - MUSCLE          1.04    1.02      0.29     4.34    2.51                    TESTES-R       603.01   58.49        0.82     2.05    2.25                    TESTES-L        44.65    2.08        0.93     2.11    2.32                  ______________________________________                                    

6.2. Abtides Binding to a Breast Cancer Antigen 6.2.1 Identification andIsolation of Abtides Binding to Polymorphic Epithelial Mucin (PEM)

The monoclonal antibody SM-3 that specifically binds the polymorphicepithelial mucin (PEM) tumor antigen found on human breast cancer cellshas been shown to be specific for the epitope defined by the amino acidsequence VTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 9) (Bruchell et al.,1989, Int. J. Cancer 44:691-696). A peptide comprising this sequence wassynthesized and used to isolate abtides from TSAR peptide libraries bymethods analogous to those described above. In these experiments, thespecific TSAR libraries used were R26, D38 and DC43. See FIGS. 10-12 fordescription of these libraries. Phage bound to PEM were eluted by eitherstandard acid elution methods, stringent acid elution methods wherephage were incubated with the PEM peptide for only 10 minutes prior towashing and elution, or were eluted using excess PEM peptide. Phage fromeach library were isolated that express peptides capable of binding toPEM. The amino acid sequences of PEM binding phage are shown in Table 6.

                                      TABLE 6                                     __________________________________________________________________________    Sequences of PEM Binding Phage                                                __________________________________________________________________________    Acid Eluted                                                                     R26 Library                                                                 A15 SFMDYFFHTPEPKPAGYPNAYTDPKHPA (SEQ ID NO: 26)                                 - A54       SSSIFDYAPFSWGSAGLSNSSINVFERS (SEQ ID NO: 27)                      - A5        SASLWDALGGWTTSAVPSYPRPHQTPGR (SEQ ID NO: 28)                      - A39       SLGLPWIDVFGRSSAEPWPFGRTNLPRS (SEQ ID NO: 29)                      - A16       SVHGAFLDSFFPWAADGPHGRGRL-TSF (SEQ ID NO: 30)                      -                                                                          DC43 Library                                                                  MA-8                                                                              EEKQGGRWSTMMPRPWCHEGGCGFLYYDAMTKPKTPPIMRTAA (SEQ ID NO: 31)               MA-21     LPRPFDDASWKLRAVKESPDGCGFGSPLLFPPYSGLPTFSSCD (SEQ ID NO: 32)           - V22       GSFESARGVTCIGNHSIGAHGCGPLRSYASFNRGSGRRH (SEQ ID NO: 33)           -                                                                           D38 Library                                                                   MA-32                                                                             DQIGSRPQTTSRSISGSWWENAKTLWQQDYAFSAPNAA (SEQ ID NO: 34)                    V23       LSDAWGNFTTSYRDSAGFPSHAMTTSQGGKRNHASRFP (SEQ ID NO: 35)                 - V21       VQLDDTSPRASGQETSQSEYDARPLLSKFAIPRPWSR (SEQ ID NO: 36)            - V1        IDSSKNRISGTGYLSFPHIRHANRRHMADDSNLAPGPS (SEQ ID NO: 37)            -                                                                           Acid Eluted: "Stringent"                                                       DC43 Library                                                                 V44 WSIGTHTGPEGKFRIPCDRSGCGGTTLTHGGLNSSPTGQHERP (SEQ ID NO: 38)                     - V39       DPCEDGYWLSSVGRAGASIRGCGAIRRSSRTLTAEYSTRASNH (SEQ ID NO:         39)                                                                          - V10       GSKRSCWGTTISNYFRPVPEGCGSASSINPNTNTGRLPSLHRQ (SEQ ID NO:            40)                                                                          - V7        SSASSGCLGRAEHLDLDSVWGCGSQADMSRRYSPWYGRPRTGV (SEQ ID NO:            41)                                                                          - V4        NVMWSSSKAGIRDCSQVPPGGCGPVNRHRASPPLTPFRHGSIR (SEQ ID NO:            42)                                                                          -                                                                          D38 Library                                                                   V45 PLTSGSSSEYRNRDDCPVYKYATNCPRLNFSPSRYSPF (SEQ ID NO: 43)                    V32       GDAYGGIFSRPRQGLADSYIHASYTGKHFFRGPRPPTR (SEQ ID NO: 44)                 - V27       STCIGAEGEWKSFHNFLQCRDATSTSSSTLDPTALRFG (SEQ ID NO: 45)           - V40       YSATLWDQFGSRQVELWSNRHASSALPFASRASVLGSR (SEQ ID NO: 46)            -                                                                           Peptide Eluted                                                                 R26 Library                                                                  P24 ILGWPFLTGLGDSTVHPRGRKGTDPS (SEQ ID NO: 47)                                      - P49       SIPSFSMWLNQLGSAALPSKGNSQDRSD (SEQ ID NO: 48)                   - P26       SRDDIFTGGPLVLFRGSKTSNHDVHSMR (SEQ ID NO: 49)                      - P6        RAELVNWYEWFHVTAEAETPVINSHNMT (SEQ ID NO: 50)                      -                                                                          DC43 Library                                                                  MP-1                                                                              GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51)               MP-2       GSASSCFPNFTARGVTVGFFGCGSPAHPAAPRVLNPATDFPAP (SEQ ID NO: 52)          - MP-22      VFRRTARSSRPIGATVFPWYGCGNSNDETLPHHDSPPSFFLGA (SEQ ID NO:        53)                                                                              - MA-13      NTCWTDLFWHGLPGGDLPRDGCGLPSELTTHPSRERRDASEN (SEQ ID NO:        54)                                                                              -                                                                          D38 Library                                                                   MP-20                                                                             IDWNWLERGQHNRGYLHSEPDAKSQPTRGPRVAPNGND (SEQ ID NO: 55)                    MP-30      GRGSDMREHWPWSMPLILDQHANDPSPRAQSHYYSHPF (SEQ ID NO: 56)             __________________________________________________________________________

6.2.2. Saturation Mutagenesis of MP-1 and Identification of AdditionalPEM Binding Abtides

A saturation mutagenesis library based on one of the PEM abtides, MP-1,was constructed. Nucleotide sequences encoding the MP-1 abtide weresynthesized using a doping scheme similar to that described in Section5.3 whereby each nucleotide was contaminated with 9% of each of theother 3 nucleotides (e.g. G=73% G, 9% A, 9% T, 9% C). The resultingmutagenic oligonucleotides were used to construct a library by TSARlibrary methods described above (see FIG. 13).

The resulting library was screened to identify phage expressing abtidescapable of binding to PEM. The binding of isolated phage to PEM wasconfirmed by an ELISA assay. Phage that were shown to bind to PEM aswell as phage that did not bind to PEM were sequenced to determine theamino acid sequences of the expressed abtides. Table 7 shows the aminoacid sequences of these positive and negative binding phage.

                                      TABLE 7                                     __________________________________________________________________________    Sequence Comparison: MP-1 Binding Motif                                       __________________________________________________________________________    Positive Binding Sequences                                                    MP1 GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51)               E4   VSTGWSGTPRWCAPGGKQGSGCGNGPRWTTLTPDLGGTRKYGP (SEQ ID NO: 57)                 - E7   GAPLWCEKLSGTGSGGFKWPGCGSGPTYNTFTPARVGSDNKWP (SEQ ID NO: 58)           - E16  GPPVWSAKSRWTGTGVLNWPGCGKVPSCSTYTPSRDRSRKSDP (SEQ ID NO: 59)            - E21  GSALLTSKGCVRGPGGLMRPGCGNDRLGKSSTYAHGGWIKTGP (SEQ ID NO: 60)            - E33  GSPVWSGDNRWRGSSPLKRPGCGNGAKCNTLKDNRKDSRKTKH (SEQ ID NO: 61)            - E44  G PLLPGEAAVHGARGLMRSGCGNGPTWNRLTAACRDSRNKGP (SEQ ID NO: 62)            - E65  GSPVWMGSTRWTGHGWFRSQGCGNVPRTNSCAPAGKDSQNKGP (SEQ ID NO: 63)            - E73  GAPVWRGNRWCSDNGELERPGCGYGPRFNILPPGRGNSRKPSP (SEQ ID NO: 64)            - E84  GSSGWKVKHRCGGPGTLQRPGCGNLPLGHTFPPTRGGSHMEGA (SEQ ID NO: 65)            - E85  GPRSWMGQPRGSDAGSCKWAGCGDAPMWRASTPGHGGPPNRGS (SEQ ID NO: 66)            - E88  EALVCRGKPPWSGPAGLLWQGCGTGPVSRTFTSAQGRSRNKTS (SEQ ID NO: 67)            - E90  GAPVVGDILWCSGARGAKWPGCGKGPTNKTFSHSRGGTQKSGL (SEQ ID NO: 68)            - E22  GAPVSRCKPACGGFWGVNWPGCGNASMCKTFTNGHGVSSDNGH (SEQ ID NO: 69)            - E29  GAHGYKNGSTCTGLGGWRCRGCQKGAMCNNPSPAGGAYHNQGP (SEQ ID NO: 70)            - E62  G PQGSEHQCCSGHWGLKFPGCGNGPICNNFTALRGASRKNGP (SEQ ID NO: 71)            - E64  GEPVWCRHSGGRVQGGLDWLGCGDGPLRYTVTPARGGPSKHGP (SEQ ID NO: 72)            - E66  GLSLVRGDSWGSGAGGWKRHGCGHGPMYNPQTPARGGSCTRNT (SEQ ID NO: 73)            - E67  VSRAWSGKPRLMGSHGLNCPGCGKGHSGIMFIPDPAGSANTPP (SEQ ID NO: 74)            - E68  CAPMWSGKPPWCVGGGVKFRGCGNRPDCNIITPRLVESRDKAL (SEQ ID NO: 75)            - E70  ADPVCSRKPDGGGLRGLRWPGCGKGPILYNVTAARGGSRNNGP (SEQ ID NO: 76)            -                                                                           Negative Binding Sequences                                                    MPI GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51)               E3   GTRVPPGFALRGGRDGLSWAGCGKAPISKTYTSARGRSRKKGS (SEQ ID NO: 77)                 - E15  RSAVSEGKPREIVPGGCMWPGCGNGRKSNTLTHGPEQFQEIEP (SEQ ID NO: 78)           - E24  SSGVGNGKPRSWAPDALNGGCGNIQFANTITPDRGGSCNQTL (SEQ ID NO: 79)             - E27  GSSVCGGQPSGRGFGGLPGPGCGNGPTSNTLTSARGGFPNKGL (SEQ ID NO: 80)            - E37  GAPLWQGDPADEVLGGSMIPGCGIGALSQTFTPTPGGSRKNVT (SEQ ID NO: 81)            - E43  AGRELRQDEGEGGAGADVARLREGPICSTFTPARGGSCPSGL (SEQ ID NO: 82)             - E49  QARVSMAISCRSGPSDLMHQGCGYGPRCNPDTTDSGGSHTNTP (SEQ ID NO: 83)            - E60  GDPECRGKPRGRWTGSLACTGCGNGPNSKICTRARGVSRNKGP (SEQ ID NO: 84)            - E72  STPGCSGYSGSGDPRCLTCTACGNGHTRKTLTPAHGRSTHKEP (SEQ ID NO: 85)            - E34  GQPECRITSGCCGTDGNKWLGCGKVDMCNTLNPAVGCHGTNGS (SEQ ID NO: 86)            - E83  REPVVGGKPWCRGPGGLRWRGCGKSQFDKIITLSRDNRRDKRP (SEQ ID NO: 23)          __________________________________________________________________________

When the sequences shown in Table 7 are compared (see particularly theamino acid residues marked in boldface type), it is possible todetermine the influence of particular amino acid residues at specificpositions in the sequence on a peptide's ability to bind to PEM. Abtidesthat bind to PEM can be characterized by the formula:

    __________________________________________________________________________    R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6 R.sub.7 R.sub.8 R.sub.9       R.sub.10 R.sub.11 R.sub.12 R.sub.13 R.sub.14 R.sub.15 R.sub.16 R.sub.17       R.sub.18 R.sub.19 R.sub.20 R.sub.21 R.sub.22 R.sub.23 R.sub.24 R.sub.25       R.sub.26 R.sub.27 R.sub.28 R.sub.29                                              - R.sub.30 R.sub.31 R.sub.32 R.sub.33 R.sub.34 R.sub.35 R.sub.36           R.sub.37 R.sub.38 R.sub.39 R.sub.40 R.sub.41 R.sub.42 R.sub.43 (SEQ ID        NO: 24)                                                                       __________________________________________________________________________

where:

R₁ =G, C, E, or V, preferably G;

R₂ =A, S, P, or L, preferably A;

R₃ =P, T, H, or L, preferably P;

R₄ =L, M, Q, G, A, or S;

R₅ =W or Y, preferably W;

R₆ =S, C, K or T, preferably S;

R₇ =E, S, C, D, V, or R;

R₈ =N, H, K, S, or E;

R₉ =L, H, R, N, Q, T, or G;

R₁₀ =W, P, R, T, or D, preferably W;

R₁₁ =W, C, V, L, or G, preferably W;

R₁₂ =S, T, M, or H, preferably S or T;

R₁₃ =G;

R₁₄ =S, A, G, N, Q, or H, preferably S;

R₁₅ =W, H, G, A, or R;

R₁₆ =G, T, E, P, V, or W, preferably G;

R₁₇ =V, F, W, K, or A;

R₁₈ =K, Q, D, E, R, or L, preferably K;

R₁₉ =R, F, or S, preferably R;

R₂₀ =P, S, I or H, preferably P;

R₂₁ =G;

R₂₂ =C;

R₂₃ =G;

R₂₄ =D, S, T, N, or H;

R₂₅ =G, D, L, or R;

R₂₆ =P or S, preferably P;

R₂₇ =M, S, D, I, L, or R;

R₂₈ =G, W, C, L, F, Y, or T, preferably G or W;

R₂₉ =S, N, V, F, H, or R;

R₃₀ =N, A, S, M, or R, preferably N;

R₃₁ =F, Q, P, or V, preferably F;

R₃₂ =S, V, I, K, A, or S;

R₃₃ =P, A, N, or Y, preferably P;

R₃₄ =G, N, or L;

R₃₅ =K, R, C, Q or L, preferably K or R;

R₃₆ =V, K, R, or A;

R₃₇ =G, D, A, or E, preferably G;

R₃₈ =S, T, P, Y or W; preferably S;

R₃₉ =R, I, L, P, A or S;

R₄₀ =N, K, or M, preferably N or K;

R₄₁ =S, R, T, E, Q, P, Y or H;

R₄₂ =G, A, S, D, N, P, Y, or K, preferably G;

R₄₃ =P, H or A.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 103                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Gly Ile Ile Asn Ala Asn Asp Pro Leu Pro Ph - #e Trp Phe Met Ser        Pro                                                                             1               5   - #                10  - #                15              - - Tyr Thr Pro Gly Pro Ala Pro Ile Asp Ile As - #n Ala Ser Arg Ala Leu                  20      - #            25      - #            30                   - - Val Ser Asn Glu Ser Gly                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Asp Leu Ser Arg Asn Leu Asp Phe Gly Arg Ph - #e Leu Leu Tyr Asn Ala      1               5   - #                10  - #                15               - - Tyr Val Pro Gly Phe Thr Pro Thr Phe Ile Se - #r Leu Thr Ala Glu His                  20      - #            25      - #            30                   - - Leu Ser Ser Pro Lys Gly                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Cys Gly Arg Ala Tyr Cys Leu Ser Gly Asn Ty - #r Asn Ile Phe Gly Ala      1               5   - #                10  - #                15               - - Leu Phe Pro Gly Val Ser Thr Pro Tyr Ala As - #p Val Gly His Asp Asp                  20      - #            25      - #            30                   - - Ala Gln Ser Trp Arg Arg                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Arg Cys Ser Pro Ile Trp Gly Ile Ser Tyr Pr - #o Phe Gly Leu Leu Ser      1               5   - #                10  - #                15               - - Ser Asn Pro Gly Val Cys His Ser Ser Asp Al - #a Glu Thr Asn Ile Arg                  20      - #            25      - #            30                   - - Asn Asp Ile Leu Thr Thr                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Gly His Ser Asn Tyr Cys Phe Val Ser Thr Le - #u Gly Met Pro Ile Val      1               5   - #                10  - #                15               - - Gly Phe Pro Ser Ile Asn Ala Arg Gly Leu Il - #e His Tyr Gly Gly Ser                  20      - #            25      - #            30                   - - Asp Pro Arg Leu Ala Ala                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Trp Gln Gly Thr His Phe Pro Tyr Thr                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Leu Val Ser Lys Asn Asp Ser Gly                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gly Ser Asp Asn Lys Ser Val Leu                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pr - #o Gly Ser Thr Ala Pro      1               5   - #                10  - #                15               - - Pro Ala His Gly Val Thr Ser Ala Pro Asp Th - #r Arg                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Tyr Xaa Xaa Leu His                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Ser Cys Val Ser His Met Leu Asp Thr Ser Ar - #g Val Tyr Thr Ala Tyr      1               5   - #                10  - #                15               - - Ala Asn Pro Gly Met Tyr Ser Arg Leu His Se - #r Pro Ala Val Arg Pro                  20      - #            25      - #            30                   - - Leu Thr Gln Ser Ser Ala                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Ser Val Gln Phe Lys Ser Ile Ser Ser Arg Se - #r Met Asp Asp Val Val      1               5   - #                10  - #                15               - - Lys Asp Pro Gly Pro Lys Pro Ala Met Tyr As - #n Arg Leu His Ser Lys                  20      - #            25      - #            30                   - - Asn Pro Phe Thr Leu Ser                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Tyr Phe Asp His Thr Tyr Ser Gly Pro Val Va - #l Lys Asn Gly Gly Leu      1               5   - #                10  - #                15               - - Val Ser Pro Gly Val Leu Ser Met Tyr Asn Ar - #g Leu His Ser Asp Gly                  20      - #            25      - #            30                   - - Gly Pro Ser Leu Ala Ser                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Thr Val Ala Thr Met His Asp Arg Leu His Se - #r Ala Pro Gly Ser Gly      1               5   - #                10  - #                15               - - Asn Leu Pro Gly Ser Tyr Asp Ile Lys Pro Il - #e Phe Lys Ala Gln Ser                  20      - #            25      - #            30                   - - Gly Ala Leu His Ser Thr                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Ile Asp Met Pro Gln Thr Ala Ser Thr Met Ty - #r Asn Met Leu His Arg      1               5   - #                10  - #                15               - - Asn Glu Pro Gly Gly Arg Lys Leu Ser Pro Pr - #o Ala Asn Asp Met Pro                  20      - #            25      - #            30                   - - Pro Ala Leu Leu Lys Arg                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Arg Leu Gly Asn His Val Trp Arg Glu Gly Gl - #y Gly Met Tyr Gln Gln      1               5   - #                10  - #                15               - - Leu His His Asn Phe Pro                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Arg Asp Ser Ala Val Glu Asn Pro Ser Val Gl - #y Gly Glu Ile Pro Met      1               5   - #                10  - #                15               - - Tyr Arg Tyr Leu His Gln Arg                                                          20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Pro Val Gln Lys Glu Tyr Gly Phe Gly Met Se - #r Gly Ala Ser Met Ile      1               5   - #                10  - #                15               - - Arg Leu Leu Arg Glu Thr Pro                                                          20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Gln Lys Gly Gly Pro Gly Leu Leu Leu Tyr Gl - #y Gly Asp Ser Met Tyr      1               5   - #                10  - #                15               - - Ile Thr Leu His Glu Pro Gly                                                          20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Leu Tyr Ala Asn Pro Gly Met Tyr Ser Arg Le - #u His Ser Pro Ala          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Gly Met Tyr Ser Arg Leu His Ser Pro Ala                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - His Cys Pro Pro Thr Pro Glu Thr Ser Cys Al - #a Thr Gln Thr Ile Thr      1               5   - #                10  - #                15               - - Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Ph - #e Leu Leu Val Ile Pro                  20      - #            25      - #            30                   - - Phe Asp Cys                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Arg Glu Pro Val Val Gly Gly Lys Pro Trp Cy - #s Arg Gly Pro Gly Gly      1               5   - #                10  - #                15               - - Leu Arg Trp Arg Gly Cys Gly Lys Ser Gln Ph - #e Asp Lys Ile Ile Thr                  20      - #            25      - #            30                   - - Leu Ser Arg Asp Asn Arg Arg Asp Lys Arg Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Cys, Glu, or Val"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ala, Ser, Pro or Leu"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Pro, Thr, His or Leu"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Leu, Met, Gln, Gly, Ala, or Ser"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Trp or Tyr"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Cys, Lys or Thr"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Glu, Ser, Cys, Asp, Val, or Arg"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Asn, His, Lys, Ser or Glu"                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Leu, His, Arg, Asn, Gln, Thr or - #Gly"        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Trp, Pro, Arg, Thr, or Asp"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Trp, Cys, Val, Leu, or Gly"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Thr, Met, or His"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly"                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Ala, Gly, Asn, Gln, or His"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Trp, His, Gly, Ala, or Arg"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Thr, Glu, Pro, Val, or Trp"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Val, Phe, Trp, Lys, or Ala"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Lys, Gln, Asp, Glu, Arg, and Leu"              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Arg, Phe, or Ser"                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Pro, Ser, Ile, or His"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 21                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly"                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 22                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Cys"                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 23                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly"                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 24                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Asp, Ser, Thr, or Asn"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 25                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Asp, or Leu"                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 26                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Pro or Ser"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 27                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Met, Ser, Asp, Ile, Leu, or Arg"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 28                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Trp, Cys, Leu, Phe, Tyr, or - #Thr"       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 29                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Asn, Val, Phe, His or Arg"                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 30                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Asn, Arg, Ser, Met, or Arg"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 31                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Phe, Gln, Pro, or Val"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 32                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Val, Ile, Lys, Ala or Ser"                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 33                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Pro, Ala, Asn, or Tyr"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 34                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Asn, or Leu"                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 35                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Lys, Arg, Cys, Gln, or Leu"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 36                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Val, Lys, Arg, or Ala"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 37                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Asp, Ala, or Glu"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 38                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Thr, Pro, Tyr, or Trp"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 39                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Arg, Ile, Leu, Pro, Ala, or Ser"               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 40                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Asn, Lys, or Met"                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 41                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Arg, Thr, Glu, Gln, Pro, Tyr, - #       or His"                                                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 42                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Gly, Ala, Ser, Asp, Asn, Pro, Tyr, - #      or Lys"                                                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 43                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Pro, His, or Ala"                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                             1               5   - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa                  20      - #            25      - #            30                   - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser or Arg"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Pro, Thr, or Ala"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Val, Ala, Asp, Glu, or Gly"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Ala      1               5   - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Ser Arg                      20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Ser Phe Met Asp Tyr Phe Phe His Thr Pro Gl - #u Pro Lys Pro Ala Gly      1               5   - #                10  - #                15               - - Tyr Pro Asn Ala Tyr Thr Asp Pro Lys His Pr - #o Ala                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Ser Ser Ser Ile Phe Asp Tyr Ala Pro Phe Se - #r Trp Gly Ser Ala Gly      1               5   - #                10  - #                15               - - Leu Ser Asn Ser Ser Ile Asn Val Phe Glu Ar - #g Ser                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Ser Ala Ser Leu Trp Asp Ala Leu Gly Gly Tr - #p Thr Thr Ser Ala Val      1               5   - #                10  - #                15               - - Pro Ser Tyr Pro Arg Pro His Gln Thr Pro Gl - #y Arg                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Ser Leu Gly Leu Pro Trp Ile Asp Val Phe Gl - #y Arg Ser Ser Ala Glu      1               5   - #                10  - #                15               - - Pro Trp Pro Phe Gly Arg Thr Asn Leu Pro Ar - #g Ser                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Ser Val His Gly Ala Phe Leu Asp Ser Phe Ph - #e Pro Trp Ala Ala Asp      1               5   - #                10  - #                15               - - Gly Pro His Gly Arg Gly Arg Leu Thr Ser Ph - #e                                      20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Glu Glu Lys Gln Gly Gly Arg Trp Ser Thr Me - #t Met Pro Arg Pro Trp      1               5   - #                10  - #                15               - - Cys His Glu Gly Gly Cys Gly Phe Leu Tyr Ty - #r Asp Ala Met Thr Lys                  20      - #            25      - #            30                   - - Pro Lys Thr Pro Pro Ile Met Arg Thr Ala Al - #a                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Leu Pro Arg Pro Phe Asp Asp Ala Ser Trp Ly - #s Leu Arg Ala Val Lys      1               5   - #                10  - #                15               - - Glu Ser Pro Asp Gly Cys Gly Phe Gly Ser Pr - #o Leu Leu Phe Pro Pro                  20      - #            25      - #            30                   - - Tyr Ser Gly Leu Pro Thr Phe Ser Ser Cys As - #p                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Gly Ser Phe Glu Ser Ala Arg Gly Val Thr Cy - #s Ile Gly Asn His Ser      1               5   - #                10  - #                15               - - Ile Gly Ala His Gly Cys Gly Pro Leu Arg Se - #r Tyr Ala Ser Phe Asn                  20      - #            25      - #            30                   - - Arg Gly Ser Gly Arg Arg His                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Asp Gln Ile Gly Ser Arg Pro Gln Thr Thr Se - #r Arg Ser Ile Ser Gly      1               5   - #                10  - #                15               - - Ser Trp Trp Glu Asn Ala Lys Thr Leu Trp Gl - #n Gln Asp Tyr Ala Phe                  20      - #            25      - #            30                   - - Ser Ala Pro Asn Ala Ala                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Leu Ser Asp Ala Trp Gly Asn Phe Thr Thr Se - #r Tyr Arg Asp Ser Ala      1               5   - #                10  - #                15               - - Gly Phe Pro Ser His Ala Met Thr Thr Ser Gl - #n Gly Gly Lys Arg Asn                  20      - #            25      - #            30                   - - His Ala Ser Arg Phe Pro                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Val Gln Leu Asp Asp Thr Ser Pro Arg Ala Se - #r Gly Gln Glu Thr Ser      1               5   - #                10  - #                15               - - Gln Ser Glu Tyr Asp Ala Arg Pro Leu Leu Se - #r Lys Phe Ala Ile Pro                  20      - #            25      - #            30                   - - Arg Pro Trp Ser Arg                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Ile Asp Ser Ser Lys Asn Arg Ile Ser Gly Th - #r Gly Tyr Leu Ser Phe      1               5   - #                10  - #                15               - - Pro His Ile Arg His Ala Asn Arg Arg His Me - #t Ala Asp Asp Ser Asn                  20      - #            25      - #            30                   - - Leu Ala Pro Gly Pro Ser                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Trp Ser Ile Gly Thr His Thr Gly Pro Glu Gl - #y Lys Phe Arg Ile Pro      1               5   - #                10  - #                15               - - Cys Asp Arg Ser Gly Cys Gly Gly Thr Thr Le - #u Thr His Gly Gly Leu                  20      - #            25      - #            30                   - - Asn Ser Ser Pro Thr Gly Gln His Glu Arg Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Asp Pro Cys Glu Asp Gly Tyr Trp Leu Ser Se - #r Val Gly Arg Ala Gly      1               5   - #                10  - #                15               - - Ala Ser Ile Arg Gly Cys Gly Ala Ile Arg Ar - #g Ser Ser Arg Thr Leu                  20      - #            25      - #            30                   - - Thr Ala Glu Tyr Ser Thr Arg Ala Ser Asn Hi - #s                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Gly Ser Lys Arg Ser Cys Trp Gly Thr Thr Il - #e Ser Asn Tyr Phe Arg      1               5   - #                10  - #                15               - - Pro Val Pro Glu Gly Cys Gly Ser Ala Ser Se - #r Ile Asn Pro Asn Thr                  20      - #            25      - #            30                   - - Asn Thr Gly Arg Leu Pro Ser Leu His Arg Gl - #n                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - Ser Ser Ala Ser Ser Gly Cys Leu Gly Arg Al - #a Glu His Leu Asp Leu      1               5   - #                10  - #                15               - - Asp Ser Val Trp Gly Cys Gly Ser Gln Ala As - #p Met Ser Arg Arg Tyr                  20      - #            25      - #            30                   - - Ser Pro Trp Tyr Gly Arg Pro Arg Thr Gly Va - #l                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - Asn Val Met Trp Ser Ser Ser Lys Ala Gly Il - #e Arg Asp Cys Ser Gln      1               5   - #                10  - #                15               - - Val Pro Pro Gly Gly Cys Gly Pro Val Asn Ar - #g His Arg Ala Ser Pro                  20      - #            25      - #            30                   - - Pro Leu Thr Pro Phe Arg His Gly Ser Ile Ar - #g                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - Pro Leu Thr Ser Gly Ser Ser Ser Glu Tyr Ar - #g Asn Arg Asp Asp Cys      1               5   - #                10  - #                15               - - Pro Val Tyr Lys Tyr Ala Thr Asn Cys Pro Ar - #g Leu Asn Phe Ser Pro                  20      - #            25      - #            30                   - - Ser Arg Tyr Ser Pro Phe                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - Gly Asp Ala Tyr Gly Gly Ile Phe Ser Arg Pr - #o Arg Gln Gly Leu Ala      1               5   - #                10  - #                15               - - Asp Ser Tyr Ile His Ala Ser Tyr Thr Gly Ly - #s His Phe Phe Arg Gly                  20      - #            25      - #            30                   - - Pro Arg Pro Pro Thr Arg                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - Ser Thr Cys Ile Gly Ala Glu Gly Glu Trp Ly - #s Ser Phe His Asn Phe      1               5   - #                10  - #                15               - - Leu Gln Cys Arg Asp Ala Thr Ser Thr Ser Se - #r Ser Thr Leu Asp Pro                  20      - #            25      - #            30                   - - Thr Ala Leu Arg Phe Gly                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - Tyr Ser Ala Thr Leu Trp Asp Gln Phe Gly Se - #r Arg Gln Val Glu Leu      1               5   - #                10  - #                15               - - Trp Ser Asn Arg His Ala Ser Ser Ala Leu Pr - #o Phe Ala Ser Arg Ala                  20      - #            25      - #            30                   - - Ser Val Leu Gly Ser Arg                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - Ile Leu Gly Trp Pro Phe Leu Thr Gly Leu Gl - #y Asp Ser Thr Val His      1               5   - #                10  - #                15               - - Pro Arg Gly Arg Lys Gly Thr Asp Pro Ser                                              20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - Ser Ile Pro Ser Phe Ser Met Trp Leu Asn Gl - #n Leu Gly Ser Ala Ala      1               5   - #                10  - #                15               - - Leu Pro Ser Lys Gly Asn Ser Gln Asp Arg Se - #r Asp                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - Ser Arg Asp Asp Ile Phe Thr Gly Gly Pro Le - #u Val Leu Phe Arg Gly      1               5   - #                10  - #                15               - - Ser Lys Thr Ser Asn His Asp Val His Ser Me - #t Arg                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - Arg Ala Glu Leu Val Asn Trp Tyr Glu Trp Ph - #e His Val Thr Ala Glu      1               5   - #                10  - #                15               - - Ala Glu Thr Pro Val Ile Asn Ser His Asn Me - #t Thr                                  20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - Gly Ala Pro Val Trp Arg Gly Asn Pro Arg Tr - #p Arg Gly Pro Gly Gly      1               5   - #                10  - #                15               - - Phe Lys Trp Pro Gly Cys Gly Asn Gly Pro Me - #t Cys Asn Thr Phe Thr                  20      - #            25      - #            30                   - - Pro Ala Arg Gly Gly Ser Arg Asn Asn Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - Gly Ser Ala Ser Ser Cys Phe Pro Asn Phe Th - #r Ala Arg Gly Val Thr      1               5   - #                10  - #                15               - - Val Gly Phe Phe Gly Cys Gly Ser Pro Ala Hi - #s Pro Ala Ala Pro Arg                  20      - #            25      - #            30                   - - Val Leu Asn Pro Ala Thr Asp Phe Pro Ala Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - Val Phe Arg Arg Thr Ala Arg Ser Ser Arg Pr - #o Ile Gly Ala Thr Val      1               5   - #                10  - #                15               - - Phe Pro Trp Tyr Gly Cys Gly Asn Ser Asn As - #p Glu Thr Leu Pro His                  20      - #            25      - #            30                   - - His Asp Ser Pro Pro Ser Phe Phe Leu Gly Al - #a                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - Asn Thr Cys Trp Thr Asp Leu Phe Trp His Gl - #y Leu Pro Gly Gly Asp      1               5   - #                10  - #                15               - - Leu Pro Arg Asp Gly Cys Gly Leu Pro Ser Gl - #u Leu Thr Thr His Pro                  20      - #            25      - #            30                   - - Ser Arg Glu Arg Arg Asp Ala Ser Glu Asn                                          35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - Ile Asp Trp Asn Trp Leu Glu Arg Gly Gln Hi - #s Asn Arg Gly Tyr Leu      1               5   - #                10  - #                15               - - His Ser Phe Pro Asp Ala Lys Ser Gln Pro Th - #r Arg Gly Pro Arg Val                  20      - #            25      - #            30                   - - Ala Pro Asn Gly Asn Asp                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - Gly Arg Gly Ser Asp Met Arg Glu His Trp Pr - #o Trp Ser Met Pro Leu      1               5   - #                10  - #                15               - - Ile Leu Asp Gln His Ala Asn Asp Pro Ser Pr - #o Arg Ala Gln Ser His                  20      - #            25      - #            30                   - - Tyr Tyr Ser His Pro Phe                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - Val Ser Thr Gly Trp Ser Gly Thr Pro Arg Tr - #p Cys Ala Pro Gly Gly      1               5   - #                10  - #                15               - - Lys Gln Gly Ser Gly Cys Gly Asn Gly Pro Ar - #g Trp Thr Thr Leu Thr                  20      - #            25      - #            30                   - - Pro Asp Leu Gly Gly Thr Arg Lys Tyr Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - Gly Ala Pro Leu Trp Cys Glu Lys Leu Ser Gl - #y Thr Gly Ser Gly Gly      1               5   - #                10  - #                15               - - Phe Lys Trp Pro Gly Cys Gly Ser Gly Pro Th - #r Tyr Asn Thr Phe Thr                  20      - #            25      - #            30                   - - Pro Ala Arg Val Gly Ser Asp Asn Lys Trp Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - Gly Pro Pro Val Trp Ser Ala Lys Ser Arg Tr - #p Thr Gly Thr Gly Val      1               5   - #                10  - #                15               - - Leu Asn Trp Pro Gly Cys Gly Lys Val Pro Se - #r Cys Ser Thr Tyr Thr                  20      - #            25      - #            30                   - - Pro Ser Arg Asp Arg Ser Arg Lys Ser Asp Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - Gly Ser Ala Leu Leu Thr Ser Lys Gly Cys Va - #l Arg Gly Pro Gly Gly      1               5   - #                10  - #                15               - - Leu Met Arg Pro Gly Cys Gly Asn Asp Arg Le - #u Gly Lys Ser Ser Thr                  20      - #            25      - #            30                   - - Tyr Ala His Gly Gly Trp Ile Lys Thr Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - Gly Ser Pro Val Trp Ser Gly Asp Asn Arg Tr - #p Arg Gly Ser Ser Pro      1               5   - #                10  - #                15               - - Leu Lys Arg Pro Gly Cys Gly Asn Gly Ala Ly - #s Cys Asn Thr Leu Lys                  20      - #            25      - #            30                   - - Asp Asn Arg Lys Asp Ser Arg Lys Thr Lys Hi - #s                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - Gly Pro Leu Leu Pro Gly Glu Ala Ala Val Hi - #s Gly Ala Arg Gly Leu      1               5   - #                10  - #                15               - - Met Arg Ser Gly Cys Gly Asn Gly Pro Thr Tr - #p Asn Arg Leu Thr Ala                  20      - #            25      - #            30                   - - Ala Cys Arg Asp Ser Arg Asn Lys Gly Pro                                          35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - Gly Ser Pro Val Trp Met Gly Ser Thr Arg Tr - #p Thr Gly His Gly Trp      1               5   - #                10  - #                15               - - Phe Arg Ser Gln Gly Cys Gly Asn Val Pro Ar - #g Thr Asn Ser Cys Ala                  20      - #            25      - #            30                   - - Pro Ala Gly Lys Asp Ser Gln Asn Lys Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - Gly Ala Pro Val Trp Arg Gly Asn Arg Trp Cy - #s Ser Asp Asn Gly Glu      1               5   - #                10  - #                15               - - Leu Glu Arg Pro Gly Cys Gly Tyr Gly Pro Ar - #g Phe Asn Ile Leu Pro                  20      - #            25      - #            30                   - - Pro Gly Arg Gly Asn Ser Arg Lys Pro Ser Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - Gly Ser Ser Gly Trp Lys Val Lys His Arg Cy - #s Gly Gly Pro Gly Thr      1               5   - #                10  - #                15               - - Leu Gln Arg Pro Gly Cys Gly Asn Leu Pro Le - #u Gly His Thr Phe Pro                  20      - #            25      - #            30                   - - Pro Thr Arg Gly Gly Ser His Met Glu Gly Al - #a                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - Gly Pro Arg Ser Trp Met Gly Gln Pro Arg Gl - #y Ser Asp Ala Gly Ser      1               5   - #                10  - #                15               - - Cys Lys Trp Ala Gly Cys Gly Asp Ala Pro Me - #t Trp Arg Ala Ser Thr                  20      - #            25      - #            30                   - - Pro Gly His Gly Gly Pro Pro Asn Arg Gly Se - #r                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - Glu Ala Leu Val Cys Arg Gly Lys Pro Pro Tr - #p Ser Gly Pro Ala Gly      1               5   - #                10  - #                15               - - Leu Leu Trp Gln Gly Cys Gly Thr Gly Pro Va - #l Ser Arg Thr Phe Thr                  20      - #            25      - #            30                   - - Ser Ala Gln Gly Arg Ser Arg Asn Lys Thr Se - #r                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - Gly Ala Pro Val Val Gly Asp Ile Leu Trp Cy - #s Ser Gly Ala Arg Gly      1               5   - #                10  - #                15               - - Ala Lys Trp Pro Gly Cys Gly Lys Gly Pro Th - #r Asn Lys Thr Phe Ser                  20      - #            25      - #            30                   - - His Ser Arg Gly Gly Thr Gln Lys Ser Gly Le - #u                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - Gly Ala Pro Val Ser Arg Cys Lys Pro Ala Cy - #s Gly Gly Phe Trp Gly      1               5   - #                10  - #                15               - - Val Asn Trp Pro Gly Cys Gly Asn Ala Ser Me - #t Cys Lys Thr Phe Thr                  20      - #            25      - #            30                   - - Asn Gly His Gly Val Ser Ser Asp Asn Gly Hi - #s                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - Gly Ala His Gly Tyr Lys Asn Gly Ser Thr Cy - #s Thr Gly Leu Gly Gly      1               5   - #                10  - #                15               - - Trp Arg Cys Arg Gly Cys Gly Lys Gly Ala Me - #t Cys Asn Asn Pro Ser                  20      - #            25      - #            30                   - - Pro Ala Gly Gly Ala Tyr His Asn Gln Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - Gly Pro Gln Gly Ser Glu His Gln Cys Cys Se - #r Gly His Trp Gly Leu      1               5   - #                10  - #                15               - - Lys Phe Pro Gly Cys Gly Asn Gly Pro Ile Cy - #s Asn Asn Phe Thr Ala                  20      - #            25      - #            30                   - - Leu Arg Gly Ala Ser Arg Lys Asn Gly Pro                                          35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - Gly Glu Pro Val Trp Cys Arg His Ser Gly Gl - #y Arg Val Gln Gly Gly      1               5   - #                10  - #                15               - - Leu Asp Trp Leu Gly Cys Gly Asp Gly Pro Le - #u Arg Tyr Thr Val Thr                  20      - #            25      - #            30                   - - Pro Ala Arg Gly Gly Pro Ser Lys His Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - Gly Leu Ser Leu Val Arg Gly Asp Ser Trp Gl - #y Ser Gly Ala Gly Gly      1               5   - #                10  - #                15               - - Trp Lys Arg His Gly Cys Gly His Gly Pro Me - #t Tyr Asn Pro Gln Thr                  20      - #            25      - #            30                   - - Pro Ala Arg Gly Gly Ser Cys Thr Arg Asn Th - #r                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - Val Ser Arg Ala Trp Ser Gly Lys Pro Arg Le - #u Met Gly Ser His Gly      1               5   - #                10  - #                15               - - Leu Asn Cys Pro Gly Cys Gly Lys Gly His Se - #r Gly Ile Met Phe Ile                  20      - #            25      - #            30                   - - Pro Asp Pro Ala Gly Ser Ala Asn Thr Pro Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - Cys Ala Pro Met Trp Ser Gly Lys Pro Pro Tr - #p Cys Val Gly Gly Gly      1               5   - #                10  - #                15               - - Val Lys Phe Arg Gly Cys Gly Asn Arg Pro As - #p Cys Asn Ile Ile Thr                  20      - #            25      - #            30                   - - Pro Arg Leu Val Glu Ser Arg Asp Lys Ala Le - #u                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                              - - Ala Asp Pro Val Cys Ser Arg Lys Pro Asp Gl - #y Gly Gly Leu Arg Gly      1               5   - #                10  - #                15               - - Leu Arg Trp Pro Gly Cys Gly Lys Gly Pro Il - #e Leu Tyr Asn Val Thr                  20      - #            25      - #            30                   - - Ala Ala Arg Gly Gly Ser Arg Asn Asn Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                              - - Gly Thr Arg Val Pro Pro Gly Phe Ala Leu Ar - #g Gly Gly Arg Asp Gly      1               5   - #                10  - #                15               - - Leu Ser Trp Ala Gly Cys Gly Lys Ala Pro Il - #e Ser Lys Thr Tyr Thr                  20      - #            25      - #            30                   - - Ser Ala Arg Gly Arg Ser Arg Lys Lys Gly Se - #r                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                              - - Arg Ser Ala Val Ser Glu Gly Lys Pro Arg Gl - #u Ile Val Pro Gly Gly      1               5   - #                10  - #                15               - - Cys Met Trp Pro Gly Cys Gly Asn Gly Arg Ly - #s Ser Asn Thr Leu Thr                  20      - #            25      - #            30                   - - His Gly Pro Glu Gln Phe Gln Glu Ile Glu Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                              - - Ser Ser Gly Val Gly Asn Gly Lys Pro Arg Se - #r Trp Ala Pro Asp Ala      1               5   - #                10  - #                15               - - Leu Asn Gly Gly Cys Gly Asn Ile Gln Phe Al - #a Asn Thr Ile Thr Pro                  20      - #            25      - #            30                   - - Asp Arg Gly Gly Ser Cys Asn Gln Thr Leu                                          35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                              - - Gly Ser Ser Val Cys Gly Gly Gln Pro Ser Gl - #y Arg Gly Phe Gly Gly      1               5   - #                10  - #                15               - - Leu Pro Gly Pro Gly Cys Gly Asn Gly Pro Th - #r Ser Asn Thr Leu Thr                  20      - #            25      - #            30                   - - Ser Ala Arg Gly Gly Phe Pro Asn Lys Gly Le - #u                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                              - - Gly Ala Pro Leu Trp Gln Gly Asp Pro Ala As - #p Glu Val Leu Gly Gly      1               5   - #                10  - #                15               - - Ser Met Ile Pro Gly Cys Gly Ile Gly Ala Le - #u Ser Gln Thr Phe Thr                  20      - #            25      - #            30                   - - Pro Thr Pro Gly Gly Ser Arg Lys Asn Val Th - #r                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:82:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                              - - Ala Gly Arg Glu Leu Arg Gln Asp Glu Gly Gl - #u Gly Gly Ala Gly Ala      1               5   - #                10  - #                15               - - Asp Val Ala Arg Leu Arg Glu Gly Pro Ile Cy - #s Ser Thr Phe Thr Pro                  20      - #            25      - #            30                   - - Ala Arg Gly Gly Ser Cys Pro Ser Gly Leu                                          35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:83:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                              - - Gln Ala Arg Val Ser Met Ala Ile Ser Cys Ar - #g Ser Gly Pro Ser Asp      1               5   - #                10  - #                15               - - Leu Met His Gln Gly Cys Gly Tyr Gly Pro Ar - #g Cys Asn Pro Asp Thr                  20      - #            25      - #            30                   - - Thr Asp Ser Gly Gly Ser His Thr Asn Thr Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:84:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                              - - Gly Asp Pro Glu Cys Arg Gly Lys Pro Arg Gl - #y Arg Trp Thr Gly Ser      1               5   - #                10  - #                15               - - Leu Ala Cys Thr Gly Cys Gly Asn Gly Pro As - #n Ser Lys Ile Cys Thr                  20      - #            25      - #            30                   - - Arg Ala Arg Gly Val Ser Arg Asn Lys Gly Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:85:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                              - - Ser Thr Pro Gly Cys Ser Gly Tyr Ser Gly Se - #r Gly Asp Pro Arg Cys      1               5   - #                10  - #                15               - - Leu Thr Cys Thr Ala Cys Gly Asn Gly His Th - #r Arg Lys Thr Leu Thr                  20      - #            25      - #            30                   - - Pro Ala His Gly Arg Ser Thr His Lys Glu Pr - #o                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:86:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                              - - Gly Gln Pro Glu Cys Arg Ile Thr Ser Gly Cy - #s Cys Gly Thr Asp Gly      1               5   - #                10  - #                15               - - Asn Lys Trp Leu Gly Cys Gly Lys Val Asp Me - #t Cys Asn Thr Leu Asn                  20      - #            25      - #            30                   - - Pro Ala Val Gly Cys His Gly Thr Asn Gly Se - #r                                  35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:87:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                              - - CTGTGCCTCG AGBNNBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN CC - #GCGG             56                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:88:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                              - - CTGTGCTCTA GAVNNVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN CC - #GCGG             56                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:89:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                              - - TCGAGBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNCCGCGG  - #                   49                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:90:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                              - - CTAGTVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNCCGCGG  - #                   49                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:91:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser or Arg"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser, Pro, Thr, or Ala"                         - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Val, Ala, Asp, Glu, or Gly"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                              - - Ser His Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa      1               5   - #                10  - #                15               - - Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa                  20      - #            25      - #            30                   - - Ser Arg Pro Ser Arg Thr                                                          35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:92:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                              - - GTGTGTCTCG AGNNNBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NB -             #NNBNNBNN     60                                                                 - - BNNBNNBNNB NNBNACGCCA N           - #                  - #                      - #81                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:93:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                              - - GTTGTGTCTA GAVNNVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NV -             #NNVNNVNT     60                                                                 - - GGCGTN                 - #                  - #                  -     #           66                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:94:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                              - - TCGAGNNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BN -             #NBNNBNNB     60                                                                 - - NNBNNBNACG CCAN              - #                  - #                      - #     74                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:95:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                              - - CTAGAVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VN -             #TGGCGTN      59                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:96:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser or Arg"                                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 25                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Tyr, His, Asn or Asp"                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 27                                                              (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ile, Met, Thr, Asn, Lys, Ser, or -          #Arg"                                                                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                              - - His Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa        Xaa                                                                             1               5   - #                10  - #                15              - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xa - #a Xaa Xaa Xaa Xaa Xaa                  20      - #            25      - #            30                   - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Se - #r Arg                              35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:97:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 82 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                              - - GTGTGTCTCG AGNNNBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NB -             #NNBNNBNN     60                                                                 - - BNNBNNBNNB NNBGGTTGTG GT           - #                  - #                     82                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:98:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                              - - GTTGTGTCTA GAVNNVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NV -             #NNVNNVNN     60                                                                 - - VNNVNNVNNV NNACCACAAC C           - #                  - #                      - #81                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:99:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                              - - TCGAGNNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BN -             #NBNNBNNB     60                                                                 - - NNBNNBGGTT GTGGT              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:100:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                             - - CTAGAVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VN -             #NVNNVNNV     60                                                                 - - NNVNNACCAC AACC              - #                  - #                      - #     74                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:101:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                               (D) OTHER INFORMATION: - #/label= Xaa                                              /note= - #"Xaa = Ser or Arg"                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                             - - His Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa      1               5   - #                10  - #                15               - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Cys Gl - #y Xaa Xaa Xaa Xaa Xaa                  20      - #            25      - #            30                   - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Ser              35          - #        40          - #        45                       - - Arg                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:102:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                             - - GGSGCSCCSG TSTGSAGSGG SAASCCSCGS TGSAGSGGSC CSGGSGGSTT SA -             #ASTGSCCS     60                                                                 - - GGCTGCGGG                - #                  - #                      - #         69                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:103:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                             - - SGGSCCSTTS TTSCGSGASC CSCCSCGSGC SGGSGTSAAS GTSTTSCASA TS -             #GGSCCSTT     60                                                                 - - CCCGCAGCC                - #                  - #                      - #         69                                                                __________________________________________________________________________

What is claimed is:
 1. A peptide comprising the amino acid sequenceWQOTHFPYT (SEQ ID NO: 6) and the amino acid sequence LVSKNDSG (SEQ IDNO: 7) that specifically binds to an antigen of human prostate carcinomacells.
 2. A peptide comprising an amino acid sequence selected from thegroup consisting of:SFMDYFFHTPEPKPAGYPNAYTDPKHPA (SEQ ID NO: 26),SSSIFDYAPFSWGSAG LSNSSINVFERS (SEQ ID NO: 27),SASLWDALGGWTTSAVPSYPRPHQTPGR (SEQ ID NO: 28),SLGLPWIDVFGRSSAEPWPFGRTNLPRS (SEQ ID NO: 29),SVHGAFLDSFFPWAADGPHGRGRLTSF (SEQ ID NO: 30),EEKQGGRWSTMMPRPWCHEGGCGFLYYDAMTKPKTPPIMRTAA (SEQ ID NO: 31),LPRPFDDASWKLRAVKESPDGCGFGSPLLFPPYSGLPTFSSCD (SEQ ID NO: 32),GSFESARGVTCIGNHSIGAHGCGPLRSYASFNRGSGRRH (SEQ ID NO: 33),DQIGSRPQTTSRSISGSWWENAKTLWQQDYAFSAPNAA (SEQ ID NO: 34),LSDAWGNFTTSYRDSAGFPSHAMTTSQGGKRNHASRFP (SEQ ID NO: 35),VQLDDTSPRASGQETSQSEYDARPLLSKFAIPRPWSR (SEQ ID NO: 36),IDSSKNRISGTGYLSFPHIRHANRRHMADDSNLAPGPS (SEQ ID NO: 37),WSIGTHTGPEGKFRIPCDRSGCGGTTLTHGGLNSSPTGQHERP (SEQ ID NO: 38),DPCEDGYWLSSVGRAGASIRGCGAIRRSSRTLTAEYSTRASNH (SEQ ID NO: 39),GSKRSCWGTTISNYFRPVPEGCGSASSINPNTNTGRLPSLHRQ (SEQ ID NO: 40),SSASSGCLGRAEHLDLDSVWGCGSQADMSRRYSPWYGRPRTGV (SEQ ID NO: 41),NVMWSSSKAGIRDCSQVPPGGCGPVNRHRASPPLTPFRHGSIR (SEQ ID NO: 42),PLTSGSSSEYRNRDDCPVYKYATNCPRLNFSPSRYSPF (SEQ ID NO: 43),GDAYGGIFSRPRQGLADSYIHASYTGKHFFRGPRPPTR (SEQ ID NO: 44),STCIGAEGEWKSFHNFLQCRDATSTSSSTLDPTALRFG (SEQ ID NO: 45),YSATLWDQFGSRQVELWSNRHASSALPFASRASVLGSR (SEQ ID NO: 46),ILGWPFLTGLGDSTVHPRGRKGTDPS (SEQ ID NO: 47), SIPSFSMWLNQLGSAALPSKGNSQDRSD(SEQ ID NO: 48), SRDDIFTGGPLVLFRGSKTSNHDVHSMR (SEQ ID NO: 49),RAELVNWYEWFHVTAEAETPVINSHNMT (SEQ ID NO: 50),GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51),GSASSCFPNFTARGVTVGFFGCGSPAHPAAPRVLNPATDFPAP (SEQ ID NO: 52),VFRRTARSSRPIGATVFPWYGCGNSNDETLPHHDSPPSFFLGA (SEQ ID NO: 53),NTCWTDLFWHGLPGGDLPRDGCGLPSELTTHPSRERRDASEN (SEQ ID NO: 54),IDWNWLERGQHNRGYLHSFPDAKSQPTRGPRVAPNGND (SEQ ID NO: 55) andGRGSDMREHWPWSMPLILDQHANDPSPRAQSHYYSHPF (SEQ ID NO: 56).
 3. A peptidecomprising an amino acid sequence selected from the group consistingof:VSTGWSGTPRWCAPGGKQGSGCGNGPRWTTLTPDLGGTRKYGP (SEQ ID NO: 57),GAPLWCEKLSGTGSGGFKWPGCGSGPTYNTFTPARVGSDNKWP (SEQ ID NO: 58),GPPVWSAKSRWTGTGVLNWPGCGKVPSCSTYTPSRDRSRKSDP (SEQ ID NO: 59),GSALLTSKGCVRGPGGLMRPGCGNDRLGKSSTYAHGGWIKTGP (SEQ ID NO: 60),GSPVWSGDNRWRGSSPLKRPGCGNGAKCNTLKDNRKDSRKTKH (SEQ ID NO: 61),GPLLPGEAAVHGARGLMRSGCGNGPTWNRLTAACRDSRNKGP (SEQ ID NO: 62),GSPVWMGSTRWTGHGWFRSQGCGNVPRTNSCAPAGKDSQNKGP (SEQ ID NO: 63),GAPVWRGNRWCSDNGELERPGCGYGPRFNILPPGRGNSRKPSP (SEQ ID NO: 64),GSSGWKVKHRCGGPGTLQRPGCGNLPLGHTFPPTRGGSHMEGA (SEQ ID NO: 65),GPRSWMGQPRGSDAGSCKWAGCGDAPMWRASTPGHGGPPNRGS (SEQ ID NO: 66)EALVCRGKPPWSGPAGLLWQGCGTGPVSRTFTSAQGRSRNKTS (SEQ ID NO: 67)GAPVVGDILWCSGARGAKWPGCGKGPTNKTFSHSRGGTQKSGL (SEQ ID NO: 68)GAPVSRCKPACGGFWGVNWPGCGNASMCKTFTNGHGVSSDNGH (SEQ ID NO: 69)GAHGYKNGSTCTGLGGWRCRGCGKGAMCNNPSPAGGAYHNQGP (SEQ ID NO: 70),GPQGSEHQCCSGHWGLKFPGCGNGPICNNFTALRGASRKNGP (SEQ ID NO: 71),GEPVWCRHSGGRVQGGLDWLGCGDGPLRYTVTPARGGPSKHGP (SEQ ID NO: 72),GLSLVRGDSWGSGAGGWKRHGCGHGPMYNPQTPARGGSCTRNT (SEQ ID NO: 73),VSRAWSGKPRLMGSHGLNCPGCGKGHSGIMFIPDPAGSANTPP (SEQ ID NO: 74),CAPMWSGKPPWCVGGGVKFRGCGNRPDCNIITPRLVESRDKAL (SEQ ID NO: 75) andADPVCSRKPDGGGLRGLRWPGCGKGPILYNVTAARGGSRNNGP (SEQ ID NO: 76).
 4. Atherapeutic or diagnostic composition comprising a peptide chosen fromthe group of peptides of claim 2 and an acceptable carrier.
 5. Atherapeutic or diagnostic composition comprising a peptide chosen fromthe group of peptides of claim 3 and an acceptable carrier.